J Protein Chem 1990 Feb;9(1):53-57
About 30% of the primary structure of acetylcholinesterase (AchE)
from
the cobra Naja naja oxiana has been determined. The sequence around the
serine residue labeled by diisopropylfluorophosphate (DFP) was found to
be TVTLFGESAGAASVGM which is similar to the active sites of AChE from
other
tissues. The part of the primary structure determined shows 76%
identity
with AChE from Torpedo and 42% identity with the Drosophila enzyme. A
surprisingly
large identity (42% in the sequence determined) was found with
lysophospholipase
from rat.
EMBO J 1990 Dec;9(12):3885-3888
Several peptides of acetylcholinesterase of Torpedo californica
labelled
with the alkylating reagent [3H]N,N-dimethyl-2-phenyl-aziridinium (DPA)
were localized within the primary structure. One peptide had the
sequence
KPQELIDVE (positions 270-278); the incorporation of DPA into this
peptide
could be specifically suppressed by propidium, which suggests that it
is
part of the peripheral anionic site. The incorporation of DPA into two
other peptides was insensitive to propidium but could be prevented by
edrophonium;
the sequence of one of the peptides assumed to be part of the anionic
site
in the catalytic centre was found to be DLFR (positions 217-220).
Decamethonium
efficiently blocked alkylation by DPA in all three investigated
peptides.
J. Prot. Chem.1990 Jun; 9(3): 362-364
No MEDLINE abstract available.
Inaugural-Dissertation am Fachbereich Chemie der Freien Universität
angefertigt am Institut für Biochemie, Arbeitsgruppe
Neurochemie,
1986-1990;
Gutachten: Prof. Hucho und Prof. Reutter, Tag der Disputation:
16.Februar
1991, Bewertung: "summa cum laude" (Bestnote)
ZUSAMMENFASSUNG:
Als Neurotransmitter-abbauendes Enzym spielt Acetylcholinesterase
(AChE)
eine Schlüsselrolle bei der chemischen Reizübertragung an der
cholinergen Synapse. Über weitere Funktionen des Proteins wird
spekuliert.
Dier Struktur der anionischen Bereiche, die für die
Substratbindung
von Bedeutung sind, ist noch nicht aufgeklärt. -
1.) Im ersten Teil der vorliegenden Arbeit wird die Reinigung von AChE
aus dem elektrischen Gewebe des Zitterrochens Torpedo californica
durch limitierte Proteolyse mit Trypsin und durch Freisetzung mit einer
spezifischen Phospholipase (PIP-LC) beschrieben. -
2.) Im zweiten Teil werden Untersuichungen zur Primärstruktur
der AChE aus dem Venom der Kobra Naja naja oxiana dargestellt
(siehe
auch Weise
et al., J Protein Chem 1990, 9:53-57): Erstmalig
wurde
eine Teilsequenz einer AChE aus einem Schlangenvenom ermittelt.
Insgesamt
wurde etwa ein Drittel des gesamten Proteins (168 Aminosäurereste)
sequenziert; die Sequenz ist zu 76 % identisch mit der
Primärstrultur
der AChE von Torpedo californica. Durch Sequenzvergleiche wurde
eine Homologie zu Lysophospho-Lipase festgestellt. Es wird gezeigt,
daß
AChE keine Lysophospholipase-Aktivität besitzt. Der aktive
Serin-Rest
im katalytischen Zentrum der AChE aus Kobra-Venom wurde mit DFP
markiert
und in der Sequenz lokalisiert; die flankierende Sequenz ist sehr
ähnlich
zu den entsprechenden Sequenzen anderer Cholinesterasen (TVTFGE-S-AGAASVGM).
3.) Der dritte Teil befaßt sich mit der Lokalisierung von
anionischen
Bindungsstellen der AChE (siehe auch Weise et al.,
EMBO
J 1990 9:3885-3888; Kreienkamp et
al.,
Proc
Natl Acad Sci U S A 1991 88:6117-6121): Mit dem
kationischen
Reagenz DPA wurden drei Peptide der AChE aus Torpedo californica
spezifisch markiert. Durch Schutzexperimente mit selektiven Liganden
konnten
diese Peptide einzelnen Bindungsstellen zugeordnet werden: Ein Peptid
der
Sequenz KPQELIDVE (Pos.270-278) gehört zu einer peripheren
anionischen
Bindungsstelle (PAS), wohingegen zwei Peptide mit den Sequenzen
SGSEMWNPN
(Pos. 79-87) und DLFR (Pos. 217-220) Teil des anionischen Subzentrums
im
katalytischen Zentrum sind. Die Reaktion von DPA mit einem
Tryptophan-Rest
bietet einen Hinweis auf die BNeteiligung von aromatischen
Aminosäure-Seitenketten
an der Bindung von kationischen Substanzen.
Proc Natl Acad Sci U S A 1991 Jul 15;88(14):6117-6121
A peptide of acetylcholinesterase (AcChoEase; acetylcholine
acetylhydrolase,
EC 3.1.1.7) from the venom of the cobra Naja naja oxiana labeled by the
affinity reagent N,N-dimethyl-2-phenylaziridinium (DPA) has been
identified.
The sequence is Gly-Ala-Glu-Met-Trp-Asn-Pro-Asn. In AcChoEase from
Torpedo
californica, a homologous peptide was labeled and isolated. Its
sequence
is Ser-Gly-Ser-Glu-Met-Trp-Asn-Pro-Asn, representing positions 79
through
87. In both cases labeling can be prevented by 0.1 mM edrophonium,
indicating
that the respective peptides form part of the anionic subsite of the
catalytic
center. The modified residue was tryptophan (Trp-84 in Torpedo
AcChoEase)
in both enzymes. In contrast to AcChoEase from Torpedo, the enzyme from
cobra venom does not contain a peripheral anionic binding site.
Trends Pharmacol Sci 1991 Nov;12(11):422-426
Acetylcholinesterase is among the most efficient enzymes known. In
order
to provide an explanation for its catalytic and regulatory mechanisms,
including the high turnover rate, the specific amino acid residues
involved
in substrate binding and hydrolysis need to be identified. In this
article,
Ferdinand Hucho, Jaak Jarv and Christoph Weise describe the topography
of the enzyme as deduced from protein chemistry studies. One result of
this approach is the finding that the binding pocket for the
substrate's
cationic cholinium group appears to be hydrophobic rather than anionic.
Multidisciplinary Approaches to Cholinesterase Functions 1992, eds: A.Shafferman and B.Velan, p.121-130, Plenum Press, New York
Proceedings of the 36th Oholo-Conference, April l992, Eilat, Israel
No MEDLINE abstract available.
Biochemistry 1992 Sep 8;31(35):8239-8244
Several photoaffinity derivatives of neurotoxin II from the venom of
the central Asian cobra Naja naja oxiana have been prepared. After
reaction
of the 125I-labeled derivatives with the nicotinic acetylcholine
receptor
from electric organ, the alpha-subunit of the nAChR is almost
exclusively
labeled by the derivative carrying the photoactivatable group in
position
Lys46. In contrast to this, a reactive group at Lys26 predominantly
labels
the gamma- and delta-subunits, while the alpha- and beta-subunits
incorporate
much less radioactivity. Competition experiments with d-tubocurarine
show
that the gamma-subunit is labeled when this derivative occupies the
high
affinity d-tubocurarine-binding site, while the delta-subunit is
labeled
by the toxin bound at the low-affinity d-tubocurarine site. A model is
discussed for the orientation of different loops of the toxin molecules
in the binding site for agonists and competitive antagonists.
Bioorg Khim 1992 Oct;18(10-11):1319-1329
Photoactivatable derivatives of the alpha-neurotoxin II from Naja
naja
oxiana are useful tools for investigating the three dimensional
architecture
of the extra-membrane part of the nicotinic acetylcholine receptor from
the electric tissue of Torpedo californica. Three derivatives, carrying
an azidobenzoyl group in position Lys-15, Lys-26, and Lys-46,
respectively,
are shown to react differently within the receptor's quaternary
structure.
Especially the Lys-26 and Lys-46 derivatives can be used for
differentiating
between the two nonequivalent alpha-subunits. The Lys-26 derivative is
applied for probing the receptor subunits next to the alpha-subunit:
the
gamma-subunit is shown to be located next to the alpha-subunit binding
d-tubocurarine with high affinity. The delta-subunit is the neighbor of
the low affinity alpha-subunit. We radioiodinated the toxin derivatives
and localized the 125I at the His-31 residue of the toxin. Very little
label was found in position Tyr-24, the only tyrosine residue of the
toxin,
or in position His-4, the only other histidine residue. This result is
important for the cleavage experiments necessary in attempts to
identify
the receptor sequence which reacted with the photolabel.
Eur J Biochem 1992 Nov 15;210(1):45-51
Starting from the finding that, for neuronal cells, the
nuclear-membrane-associated
protein kinase C (PKC) is the so-called 'membrane inserted',
constitutively
active form, we attempted to identify substrates of this nuclear PKC.
For
this purpose, nuclear membranes and other subcellular fractions were
prepared
from bovine brain, and in-vitro phosphorylation was performed. Several
nuclear membrane proteins were found, the phosphorylation of which was
inhibited by specific PKC inhibitors and effectively catalyzed by added
PKC. Combining the methods of two-dimensional gel electrophoresis,
in-situ
digestion, reverse-phase HPLC and microsequencing, two of these nuclear
PKC substrates were identified; the known PKC substrate Lamin B2, which
serves as a control of the approach and the nucleolar protein B23. Our
data suggest, that, for B23, Ser225 is a site of phosphorylation by
PKC.
Eur J Biochem 1993 May 1;213(3):1235-1242
The secondary structure of the acetylcholinesterase and its
temperature
behaviour have been investigated using Fourier-transform infrared
(FTIR)
spectroscopy. The data are compared to the structure obtained by X-ray
analysis of the crystalline enzyme. The secondary structure was
determined
using the spectral features observed in the amide-I band (H2O buffer)
and
amide-I' band (D2O buffer) at 1600-1700 cm-1, taking advantage of
resolution-enhancement
techniques along with least-squares band-fitting procedures. The
relative
amounts of different secondary-structure elements, 34-36% for
alpha-helices,
19-25% for beta-sheets, 15-16% for turns and 13-17% for irregular
structures,
were estimated. These data, obtained with the enzyme in solution,
correlate
well with X-ray data of the crystalline protein [Sussman, J. L., Hard,
M., Frolow, F., Oefner, C., Goldman, A., Toker, L. & Silman, I.
(1991)
Science 253, 872-879]. These results are also in good agreement with
those
obtained by computing the psi and phi angles of the peptide backbone
using
the Kabsch and Sanders method [Kabsch, W. & Sanders, C. (1983)
Biopolymers
22, 2577-2637]. In conjunction with the X-ray data, two bands in the
FTIR
spectra were assigned to different populations of long and short
alpha-helices.
Until now this phenomenon has only been described by theoretical
calculations
[Nevskaya, N. A. & Chirgadze, Yu. N. (1976) Biopolymers 15,
637-648].
The relationship between the thermally induced loss of enzyme activity
and secondary-structure changes has also been investigated. The
decrease
in enzyme activity to zero at 30-40 degrees C was accompanied only by
minor
changes in the secondary structure. At 55-60 degrees C, denaturation of
AChE occurs. In this temperature range, all bands assigned to the
various
secondary-structure elements abruptly disappear in a co-operative and
irreversible
manner, whereas the beta-aggregation bands (at 1622 cm-1 and the
corresponding
high-frequency band) increase in intensity at the same rate.
Bioorganitscheskaja Kimiya 1994, 20: 1047-1060
After irradiation of a complex of the nicotinic acetylcholine
receptor
(AChR9 with iodinated (Lys-Abz-26)-neurotoxin II, the labeled
delta-subunit
of AChR was isolated and cleaved with LysC endoproteinase, the
hydrolysate
being separated by rpHPLC. In a MS analysis of the radioactive
fraction,
a peptide of the delta-subunit with a mass of 2593 was detected. By
purification
of the radioactive rpHPLC fraction with the aid of electrophoresis in a
tricine gel, three radioactive bands were obtained (16, 10 and 8 kDa99:
Edman degradation yielded the sequence of a fragment starting from
Phe-148
for all of these. On further cleavage ofvthe radioactive fraction
within
the gel matrix using AspN protease, followed by rp-HPLC, radioactivity
was eluted under conditions close to those for the elution of the
radioactive
peptide 30-44 obtained by successive cleavage of the 125I-neurotoxin II
by LysC- and AspN-proteinases. This result shows the presence of the
corresponding
neurotoxin fragment in the sample in which the above-mentioned sequence
of the AChR was detected.
Since no sequences of the neurotoxin were detected in the radioactive
products of the cross-links in model experiments at the picomolar
level,
neurotoxin II and its fragments were investigated by Edman degradation
at the picomole level and so was the influence of the p-azidobenzoyl
group
and its photo-activation on the degradation process. On the whole, the
sequencing of neurotoxin II and its fragments containing photo-labeled
and iodinated residues took place with extremely low initial yields; a
further fall in the yields was observed on the degradation of
irradiated
Lys26-peptides. The results obatined explain the difficulties in the
detection
of the sequences of the neurotoxin in cross-link products available in
amounts of only 10 - 20 pmoles. For the unambiguous identification of
the
sections by which the neurotoxin is bound in the AChR subunits, further
investigations are necessary which include the study of the products of
coross-links during separation and analysis of the peptides.
Eur J Biochem 1994 Mar 15;220(3):1005-1011
All possible N-glycosylation sites of the delta subunit of the
nicotinic
acetylcholine receptor from Torpedo californica electric tissue are
utilized.
By a combination of microsequencing and mass spectrometry, it was shown
that a high-mannose-type oligosaccharide is bound at Asn143 of the
delta
subunit. The oligosaccharides at positions Asn70 and Asn208 of the
delta
subunit are probably of the complex type. The utilized glycosylation
sites
pose restrictions on possible transmembrane folding models of the
subunit.
Electrophoresis 1995 May;16(5):841-850
High resolution two-dimensional polyacrylamide gel electrophoresis
(2-D
PAGE), followed by computer-assisted image analysis (PDQUEST) was used
to screen atrial and ventricular protein patterns for quantitative and
qualitative differences in protein expression. Myocardial proteins from
left ventricular (LV) and right atrial (RA) samples from end-stage,
failing
explanted hearts and from a healthy donor heart (control) were
separated
by 2-D large gel electrophoresis. Ten RA versus ten LV gels from
explanted
dilated cardiomyopathic (DCM) hearts were analyzed for quantitative
differences
in their spot patterns. Of the 197 spots matched to every gel, 40 spots
differed significantly in intensity between RA and LV for DCM patients.
A larger number of atrial and ventricular gels (20 RA, 20 LV) from DCM
patients and from a healthy donor heart (4 RA, 4 LV gels) were analyzed
for qualitative differences in protein expression. Three protein spots
(SSP 1120: M(r)/pI:20.5 kDa/4.6; SSP 1119: M(r)/pI:20.6 kDa/4.5; SSP
0117:M(r)/pI:20.7/
< 4.5) that are present in all RA gels for DCM patients are absent
in
all LV gels. Two protein spots (SSP 0112: M(r)/pI:17.2 kDa,/ < 4.4;
SSP 0114:M(r)/pI:17.6 kDa/ < 4.4) occur only in all LV gels but not
in the RA gels. These five qualitatively differing spots are identical
in DCM patients and in the healthy donor heart. Some of the differing
spots
were internally sequenced and identified as myosin light chain isoforms
(myosin light chain 2, atrial; myosin light chain 2, ventricular;
myosin
light chain 1, atrial) with the Protein Identification Resource (PIR)
accession
numbers A44451, S03708, A30881, respectively. Additionally,
phosphoglycerate
mutase (PIR: JQ0750) and ATP synthase alpha chain (PIR: S17193) were
identified.
Thus, quantitative and qualitative differences between atrial and
ventricular
protein patterns were identified by 2-D PAGE. A characteristic
distribution
of myosin light chains between atrial and ventricular human myocardium
was found using our approach.
Eur J Biochem 1995 Mar 15;228(3):947-954
A new series of photoactivatable and cleavable derivatives of
neurotoxin
II from the cobra Naja naja oxiana is investigated which can be used
for
mapping the surface topology of the nicotinic acetylcholine receptor
from
Torpedo electric tissue. The preparation and characterisation of five
toxin
derivatives, each with a radioactive
125I-azidosalicylamidoethyl-1,3'-dithiopropyl
group in a defined position within the primary structure, are
described.
The photoinduced cross-linking reaction of the toxin derivatives with
membrane-bound
receptor is investigated. The photoactivatable group located at
position
K25 reacts almost exclusively with the delta subunit of the receptor,
whereas
the K15 derivative reacts with the alpha and beta subunits. The other
derivatives
did not react with the receptor to any significant extent. It is shown
that, with respect to the receptor subunits, the cross-linking pattern
depends on the length and chemical nature of the cross-linking group.
J Cell Biochem 1995 Oct;59(2):177-185
The treatment of isolated cell nuclei with citric acid was described
as a method for separating inner and outer nuclear membrane. Using cell
nuclei from bovine cerebral cortex, we can show that citric acid does
not
cause a separation of the two nuclear membranes, but extracts a
specific
set of proteins from the nuclei. The extraction of proteins is not just
an effect of damaging the nuclear membrane or destructing the
cytoskeleton,
but rather a specific effect of citric acid treatment. One of the
extracted
proteins, chosen as a marker for the putative outer nuclear membrane
fraction,
has an apparent molecular weight of 145 kDa and is located in the
nucleoplasm
as shown by immunofluorescence microscopy. By sequencing tryptic
peptides
it was identified as RNA helicase A, an abundant nuclear protein
assumed
to participate in the processing of mRNA.
J Cell Biol 1995 Sep;130(6):1263-1273
Nsp1p interacts with nuclear pore proteins Nup49p, Nup57p and Nic96p
in a stable complex which participates in nucleocytoplasmic transport.
An additional p80 component is associated with Nsp1p, but does not
co-purify
with tagged Nup57p, Nup49p and Nic96p. The p80 gene was cloned and
encodes
a novel essential nuclear pore protein named Nup82p.
Immunoprecipitation
of tagged Nup82p reveals that it is physically associated with a
fraction
of Nsp1p which is distinct from Nsp1p found in a complex with Nup57p,
Nic96p
and Nup49p. The Nup82 protein can be divided into at least two
different
domains both required for the essential function, but it is only the
carboxy-terminal
domain, exhibiting heptad repeats, which binds to Nsp1p. Yeast cells
depleted
of Nup82p stop cell growth and concomitantly show a defect in
poly(A)+RNA
export, but no major alterations of nuclear envelope structure and
nuclear
pore density are seen by EM. This shows that Nsp1p participates in
multiple
interactions at the NPC and thus has the capability to physically
interact
with different NPC structures.
Eur J Immunol 1995 Jun;25(6):1749-1754
TRAP is a tumor necrosis factor (TNF)-related, 33-kDa type II
transmembrane
protein almost exclusively expressed on the surface of activated CD4+ T
lymphocytes. Interaction of TRAP with CD40 on B cells is of paramount
importance
for immunoglobulin class switching and subsequent synthesis of IgG, IgA
or IgE in vivo. We now provide evidence that activated T cells not only
express cell membrane-associated TRAP but also a soluble form of TRAP
(sTRAP).
After generating monoclonal antibodies against TRAP and establishing a
TRAP-specific enzyme-linked immunosorbent assay we were able to detect
substantial amounts of sTRAP in the supernatants of activated T cells.
The onset and rate of sTRAP release was found to parallel the
expression
of TRAP on the cell surface. sTRAP, an 18-kDa protein, is generated by
proteolytic processing of full-length TRAP in an intracellular
compartment.
Starting with methionine 113 of full-length TRAP, sTRAP lacks the
transmembrane
region and a part of the extracellular domain but contains the entire
TNF-alpha
homology region and can, therefore, bind to CD40. Like other members of
the TNF superfamily (e.g. TNF-alpha, Fas/APO-1 ligand), TRAP thus has
the
potential to be biologically active not only in a transmembrane form
but
also as a soluble molecule.
Eur J Biochem 1995 Dec 1;234(2):427-430
Cross-linking an alpha-neurotoxin with a known three-dimensional
structure
and with photoactivatable groups in known positions to native
membrane-bound
acetylcholine receptor reveals its quaternary structure, including the
handedness of its circular subunit arrangement. Photolabelling with
alpha-neurotoxin
carrying the photoactivatable group at position Lys46 is inhibited by
the
competitive antagonist (+)-tubocurarine in a biphasic manner,
indicating
that it reacts with both alpha-subunits that were shown to have
different
affinities for this antagonist [Neubig, R. R. & Cohen, J. B. (1979)
Biochemistry 18, 5464-5475]. Lys46 is located on loop III of the
neurotoxin.
The other information necessary for the elucidation of the handedness
was
provided by the recent finding that the central loop of the toxin (loop
II) is oriented towards the central pore of the receptor, securing the
overall orientation of the bound toxin [Machold, J., Utkin, Y. N.,
Kirsch,
D., Kaufmann, R., Tsetlin, V. & Hucho, F. (1995b) Proc. Natl Acad.
Sci. USA 92, 7282-7286]. Looking at the receptor from the synaptic side
of the postsynaptic membrane, it was concluded that the clockwise
subunit
arrangement is alpha H-gamma-alpha L-delta-beta (alpha H and alpha L
are
the alpha-subunits binding (+)-tubocurarine with high and low affinity,
respectively). Its mirror image alpha alpha L-gamma-alpha H-beta-delta
could thus be excluded.
Insect Biochem Mol Biol 1995 Dec;25(10):1081-1091
A prophenoloxidase (PPO) was purified from the hemolymph of the
larvae
of Galleria mellonella. A 135-fold purification of the proenzyme with
25%
yield was achieved by a combination of different chromatographic
methods.
An alternative micropreparation of pure PPO by a novel method for
native
electrophoresis in polyacrylamide gel is also described. The molecular
mass of the native PPO was estimated to be 300 kDa by the pore-limit
gradient
electrophoresis in polyacrylamide gel. In the presence of sodium
dodecyl
sulphate, two closely migrating subunits of 80 and 83 kDa were detected
under non-reducing conditions. The PPO was shown to be a glycoprotein
and
its isoelectric point was 6.2. The amino-acid composition of the
purified
protein was similar to the PPO from Bombyx mori. The monospecific
antibody
raised against the purified PPO crossreacted with the
(pro)phenoloxidase
in hemolymph of Manduca sexta. The activation of the PPO with
chymotrypsin
was investigated and two proteins of 67 and 50 kDa were found to be
products
of the proteolytic cleavage. The N-terminus of the G. mellonella PPO
was
blocked, but eleven partial internal sequences were determined after
fragmentation
of the purified PPO with trypsin. Three of these peptides exhibited
significant
homology with highly conserved sequences found in arthopod hemocyanins
and insect storage proteins, which indicates that the PPO belongs to
this
family.
Cell 1996 Jan 26;84(2):265-275
In a genetic screen for nucleoporin-interacting components, a novel
nuclear pore protein Nup84p, which exhibits homology to mammalian
Nup107p,
was isolated. Nup84p forms a complex with five proteins, of which
Nup120p,
Nup85p, Sec13p, and a Sec13p homolog were identified. Upon isolation of
Sec13p-ProtA, nucleoporins were still associated, but the major
copurifying
band was a 150 kDa protein, showing that Sec13p occurs in two
complexes.
Disruption of any of the genes encoding Nup84p, Nup85p, or Nup120p
caused
defects in nuclear membrane and nuclear pore complex organization, as
well
as in poly(A)+ RNA transport. Thus, the Nup84p complex in conjunction
with
Sec13-type proteins is required for correct nuclear pore biogenesis.
Biochem Biophys Res Commun 1996 Aug 23;225(3):1078-1083
VILIP is a member of the visinin/recoverin family of neuronal
EF-hand
Ca(2+)-binding proteins. Cell fractionation revealed cytoplasmic,
membrane-
and cytoskeleton-associated pools of VILIP. The association with the
cytoskeletal
protein fraction is Ca(2+)-dependent and may be mediated by direct
interaction
with actin. This is concluded from the observations that (i)
Ca(2+)-loaded
recombinant VILIP binds actin in an overlay assay; (ii) in the presence
of Ca(2+), beta-actin co-immunoprecipitates with native VILIP from
brain
extracts, and (iii) actin and VILIP are co-localized in PC12 cells
stably
transfected with VILIP cDNA. The interaction of VILIP with the cortical
cytoskeleton through actin may facilitate a Ca(2+)-dependent
recruitment
of VILIP to the cell membrane.
J.Insect Physiology 1997, 43: 383-391.
Apolipophorin III (apoLp-III) was isolated from the haemolymph of
last
instar larvae of Galleria mellonella. The ultravilet spectrum
and
the N-terminal amino acid sequence reveal high similarities with the
apoLp-III
from Manduca sexta. The protein is heat-stable. The mass of
apoLp-III
was determined to be 18,077 Da using mass spectrometry. The heat
treatment
(90 degrees C, 30 min) resulted in a pI shift from 6.6 for the
non-heated
to 6.1 for the heat-treated protein without change in the molecular
mass,
indicating that a conformational change might have been caused by the
heat
treatment, rather than covalent alterations. Intrahaemocoelic injection
of pure apoLp-III into last instar G.mellonella larvae is followed by a
dose-dependent increase of anti-bacterial activity in cell-free
haemolymph
of treated larvae 24h after injection. Furthermore, pure apoLp-III
enhances
the phagocytic activity of isolated haemcytes in vitro.
The newly dicovered role of apoLp-III in inducing immune-related
functions
in insects is discussed in regard to the known features of this
molecule
in lipid metabloism. Arylphorin, another heat-stable protein in
G.mellonella
haemolymph, was likewise isolated in this study. The protein was
identified
by N-terminal protein sequnecing, the sequence obtained exactly matches
the known sequence for this protein.
J Protein Chem 1997 Apr;16(3):161-170
Here we report on the use of iodination of the membrane-bound
nicotinic
acetylcholine receptor (nAChR) from Torpedo californica electric tissue
in order to define surface-exposed portions of the receptor molecule.
Membrane-bound
nAChR was 125I-iodinated using the oxidation agent Iodo-Gen. The
iodinated
subunits were separated by preparative gel electrophoresis, desalted,
and
cleaved with trypsin. The resulting peptides were separated by
reverse-phase
HPLC and the radioactive peptides were identified by mass spectrometry
and protein sequencing. For the delta-subunit, we identified five
iodinated
peptides containing the tyrosine residues deltaTyr17, deltaTyr74,
deltaTyr365,
deltaTyr372, and deltaTyr428. The surface exposition of these amino
acids
is in agreement with the four-transmembrane-segment model (4TM model)
of
the nAChR, but the assignment to the intra- or extracellular surface is
doubtful. According to this model, the N-terminal portion of the
receptor
subunits including the iodinated residues deltaTyr17 and deltaTyr74 is
extracellular and deltaTyr372 as a site of tyrosine phosphorylation is
located on the cytoplasmic side. But since this latter residue is among
the first to be iodinated using an immobilized iodination agent, its
true
position with respect to the membrane bilayer is not clear.
Virus Res 1997 Jun;49(2):215-223
The observation that HIV in vitro can infect CD4- and Gal-C-negative
brain cell lines has stimulated this study to identify alternative
gp120-binding
proteins on brain cells. HIV-1 gp120 binding proteins of the
CD4-negative
and Gal-C-negative, non-productively infectable human glioblastoma cell
line D54 were purified by affinity chromatography over a
gp120-conjugated
sepharose column and identified by peptide microsequencing. The binding
capacity and specificity of this column was controlled using extracts
of
CD4-positive cells. Two of seven prominent proteins eluted from the
gp120
affinity column specifically bound gp120 in Western blot overlay
experiments
and were identified by subsequent immunoblotting and microsequencing as
ezrin and moesin, members of the ERM (ezrin, radixin, moesin) family of
cellular structural membrane proteins. Antibodies to ezrin and moesin
specifically
recognized the eluted gp120 binding proteins confirming their
identification.
Ezrin and moesin are structural proteins binding to the cellular
membrane
and to several cytoskeletal and transmembrane proteins. Our results
suggest
that ezrin and moesin might play a role as gp160/gp120 binding proteins
during the uptake, the assembly or the budding of HIV.
Dev Comp Immunol 1997 Jul;21(4):323-336
The suitability of the hemocyte cell line BTI-EA-1174-A from
Estigmene
acraea (Lepidoptera) to serve as a tool for studying insect immune
reactions
in vitro was investigated. Addition of bacterial lipopolysaccharides to
the cultures caused enhanced phagocytosis of silica beads, as well as
increased
lysozyme activity in the cell culture supernatants. Addition of fungal
beta 1,3-glucans did not result in any activation. The LPS-influenced
(1
mg/mL) increase of phagocytic reactions against the silica beads was at
its highest within 24 h after LPS-addition. Activated cells exhibited
drastic
changes in their morphology in connection with reduced cell numbers in
the cultures but without increased mortality rates. LPS-dosages higher
than 10 micrograms/mL LPS provoked significantly enhanced lysozyme
activities.
A maximal induction took place with 1 mg/mL LPS. The lysozyme activity
started to rise 2 days after LPS-addition, further increase was
observed
up to the seventh day. The responsible protein was isolated from cell
culture
supernatants and N-terminally sequenced. The exact molecular mass was
determined
by mass spectrometry as 14.080 kDa. The amino acid sequence of the
analysed
portion revealed high sequence-similarity to the lysozymes of other
lepidopteran
insects as well as to hen egg lysozyme. Further results presented in
this
paper give indications for the existence of soluble molecules which are
released by the cells and which enhance the LPS-triggered activation.
J Biol Chem 1997 Sep 26;272(39):24319-24324
N-Acetylneuraminic acid (Neu5Ac) is the precursor of sialic acids, a
group of important molecules in biological recognition systems.
Biosynthesis
of Neu5Ac is initiated and regulated by its key enzyme,
UDP-N-acetylglucosamine
2-epimerase (UDP-GlcNAc 2-epimerase, EC 5.1. 3.14)/N-acetylmannosamine
kinase (ManNAc kinase, EC 2.7.1.60) in rat liver (Hinderlich, S.,
Stasche,
R., Zeitler, R., and Reutter, W. (1997) J. Biol. Chem. 272,
24313-24318).
In the present paper we report the isolation and characterization of a
cDNA clone encoding this bifunctional enzyme. An open reading frame of
2166 base pairs encodes 722 amino acids with a predicted molecular mass
of 79 kDa. The deduced amino acid sequence contains exact matches of
the
sequences of five peptides derived from tryptic cleavage of the enzyme.
The recombinant bifunctional enzyme was expressed in COS7 cells, where
it displayed both epimerase and kinase activity. Distribution of
UDP-GlcNAc
2-epimerase/ManNAc kinase in the cytosol of several rat tissues was
investigated
by determining both specific enzyme activities. Secreting organs
(liver,
salivary glands, and intestinal mucosa) showed high specific activities
of UDP-GlcNAc 2-epimerase/ManNAc kinase, whereas significant levels of
these activities were absent from other organs (lung, kidney, spleen,
brain,
heart, skeletal muscle, and testis). Northern blot analysis revealed no
UDP-GlcNAc 2-epimerase/ManNAc kinase mRNA in the non-secreting tissues.
Protein Expr Purif 1998 Mar;12(2):226-232
A procedure for purifying the Torpedo californica nicotinic
acetylcholine
receptor subunits is proposed which involves preparative SDS-PAGE
followed
by reverse-phase HPLC on a C4 column in an acetonitrile-isopropanol
system.
By this method, the alpha-subunit can be completely separated from the
43-kDa protein which migrates very close to it on SDS-PAGE, and the
delta-subunit
can be isolated free from the beta-subunit of Na+, K(+)-ATPase
comigrating
with it on SDS-PAGE. The purity of all acetylcholine receptor subunits
thus obtained was verified by Edman degradation and MALDI
mass-spectrometric
analysis which could be performed quite easily on the HPLC-purified
samples.
In general, we observed a good correlation between the experimentally
determined
molecular masses and those calculated from the amino acid sequences and
when known, posttranslational modifications (glycosylation and
phosphorylation)
of individual receptor subunits. Transfer of the isolated receptor
subunits
into 1% octyl-beta-D-glucopyranoside generates samples suitable for
functional
studies and enzymatic proteolysis or deglycosylation.
Eur J Biochem 1998 Feb 15;252(1):133-139
N-Acetylglucosamine, a major sugar in complex carbohydrates, enters
the pathways of aminosugar metabolism by the action of
N-acetylglucosamine
kinase (EC 2.7.1.59). In this study we report the purification to
homogeneity
of GlcNAc kinase from rat liver cytosol using salmine sulfate
precipitation,
chromatography on phenyl-Sepharose, ATP-agarose and MonoQ, and finally
gel filtration on Superdex 200. It was characterized as a dimer of
39-kDa
subunits. About 25% of the amino acid sequence of GlcNAc kinase was
established
by peptide mapping. Part of the ATP-binding site of GlcNAc kinase was
identified
by sequence comparison with related hexokinases, including the
bifunctional
enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase
(EC
5.1.3.14/2.7.1.60), the key enzyme of N-acetylneuraminic acid
biosynthesis
in rat liver. The Cys residues in the active sites of GlcNAc kinase and
ManNAc kinase were characterized by chemical modification with
N-ethylmaleimide,
iodoacetamide and 5,5'-dithiobis(2-nitrobenzoic acid). The finding that
the substrates GlcNAc and ManNAc protected their respective enzymes
from
inhibition by the above sulfhydryl reagents indicates that Cys residues
are located in or near the active sites of both enzymes. Use of the
specific
dithiol-modifying chemical reagents, sodium meta-periodate, sodium
meta-arsenite/2,3-dimercaptopropanol
and diazenedicarboxylic acid bis-N,N'-dimethylamide revealed that the
active
sites of GlcNAc kinase and ManNAc kinase possess at least one pair of
vicinal
thiols. Chemical treatment of UDP-GlcNAc 2-epimerase provided no
evidence
for the presence of cysteine in the active site of this enzyme. From
the
incorporation of N-[3H]ethylmaleimide into GlcNAc kinase in the absence
and presence of ligands we estimated that the active site of GlcNAc
kinase
contains two Cys residues.
J. Prot. Chem. 1998 Oct;17(7):633-41.
The complete amino acid sequence of apolipophorin-III (apoLp-III), a
lipid-binding hemolymph protein from the greater wax moth Galleria
mellonella,
was determined by protein sequencing. The mature protein consists of
163
amino acid residues forming a protein of 18,075.5 Da. Its sequence is
similar
to apoLp-III from other Lepidopteran species, but remarkably different
from the apoLp-IIIs of insects from other orders. As shown by mass
spectrometric
analysis, the protein carries no modifications. Thus, all of its known
physiological functions, including its recently discovered immune
response
stimulating activity, must reside in the protein itself.
Eur J Biochem 1998 Mar 1;252(2):222-228
To obtain structural information on the nicotinic acetylcholine
receptor
from Torpedo electric tissue we modified and cross-linked lysine
residues
with the agonistic bifunctional reagent [14C]dimethyl suberimidate.
This
reagent labels exposed lysine residues, especially those located near
the
ligand-binding site, and cross-links lysine residues located not more
than
11 A, the length of the cross-linker, apart. Using this method, we
identified
a cross-link located between betaLys177 and betaLys191 showing that the
13 amino acids in between form a loop with these two residues located
at
the surface. Cross-linking also occurred between the vicinal lysine
residues
alphaLys76 and alphaLys77, indicating that these neighbouring lysine
residues
are not involved in a beta-sheet structure. A total of 21 out of 97
lysine
residues present in the receptor were modified by [14C]dimethyl
suberimidate.
Thus these residues are located on the accessible extramembrane
surface.
The two lysine residues alphaLys76 and alphaLys179 were predominantly
labelled.
Because of the agonistic property of [14C]dimethyl suberimidate [Watty,
A., Methfessel, C. & Hucho, F. (1997) Proc. Natl Acad. Sci. USA 94,
8202-8209] this might be due to their close proximity to the ligand
binding
site.
Eur J Biochem 1998 Aug 1;255(3):535-543
Purification and characterization of an inducible metalloprotease inhibitor from the hemolymph of greater wax moth larvae, Galleria mellonella.
Wedde, M., Weise, C., Kopácek, P., Franke, P. and Vilcinskas, A.
Institute of Zoology, Free University of Berlin, Germany.
In this paper, we report the detection, purification and
characterization
of the first metalloprotease inhibitor (IMPI) from invertebrates. IMPI
was purified from the hemolymph of last-instar larvae of Galleria
mellonella
by precipitation with trichloroacetic acid and heat followed by
affinity
chromatography on a thermolysin-Sepharose column and gel filtration or
reverse-phase high-performance liquid chromatography. For the detection
of inhibitor activity, a new azocoll assay was established. IMPI was
only
detectable in larvae that had been injected with bacterial or fungal
provocators,
suggesting that it is induced nonspecifically during the humoral immune
response. Injection of larvae with IMPI rendered them resistant to
thermolysin,
in quantities that normally would be lethal for them. IMPI was shown to
be specific for metalloproteases. The molecular mass of IMPI was
determined
by mass spectrometry to be 8360 Da. Purified IMPI was heterogeneous,
owing
to different degrees of glycosylation with hexose/hexosamine and
deoxyhexose
residues. Ten cysteine residues were found in the molecule, and these
are
presumed to form five disulfide bridges. The amino terminus was
blocked,
but a partial amino-acid sequence starting from the thermolysin
cleavage
site was determined; this sequence exhibited no similarity with other
known
proteins, suggesting that the IMPI represents a new type of protease
inhibitor.
J Protein Chem 1999 Feb;18(2):205-14
Butyrylcholinesterase is complexed with transferrin in chicken serum.
Weitnauer, E., Ebert, C., Hucho, F., Robitzki, A., Weise, C. and Layer, P.G.
Department of Developmental Biology and Neurogenetics, Darmstadt University of Technology, Germany.
The function of the enzyme butyrylcholinesterase (BChE) both in
serum
and in brain is unclear. In serum, BChE has been found complexed with
several
biomedically relevant proteins, with which it could function in
concert.
Here, the existence of a similar complex formed between BChE and
sero-transferrin
from adult chicken serum was elucidated. In order to identify both
proteins
unequivocally, we improved methods to highly purify the 81-kDa BChE and
the coisolated 75-kDa transferrin, which then allowed us to tryptically
digest and sequence the resulting peptides. The sequences as revealed
for
BChE peptides were highly identical to mammalian BChEs. A tight complex
formation between the two proteins could be established (a) since
transferrin
is coisolated along with BChE over three steps including procainamide
affinity
chromatography, while transferrin alone is not bound to this affinity
column,
and (b) since imunoprecipitation experiments of whole serum with a
transferrin-specific
antiserum allows us to detect BChE in the precipitate with the
BChE-specific
monoclonal antibody 7D11. The possible biomedical implications of a
complex
between transferrin and BChE which here has been shown to exist in
chicken
serum are briefly discussed.
J Invertebr Pathol. 1999 Sept; 74: 209-212
Institute of Zoology, Free University of Berlin, Germany.
Here we report on the isolation and sequencing of three cecropins
from
cell culture supernatants of the hemocyte line BTI-EA-1174-A (Granados
and Naughten, 1976) derived from the Lepidopteran insect Estigmene
acraea.
As in previous experiments, this work was done to ensure the
suitability
of this cell line for in vitro studies of lepidopteran immune
reactions.
- The HPLC separation of the prepurified E.acraea cell culture
supernatant
delvered seven fractions with anti-E.coli activity. The peptides
isolated
in three of these fractions were sequenced. The primary structure
obtained
are nearly identical to three variants (IIID, IIIE and IIIF) of the
so-calles
hyphancin, a cecropin isolated from the lepidopteran insect Hyphantria
cunea (Park et al., 1997).
Biochim Biophys Acta 1999 Aug 17;1433(1-2):16-26
Insect immune activation by recombinant Galleria mellonella apolipophorin III
Niere, M., Meislitzer, C., Dettloff, M., Weise, C., Ziegler, M. and Wiesner, A.
Free University of Berlin, Institute of Zoology, Konigin-Luise-Str. 1-3, D-14195, Berlin, Germany
Apolipophorin III (apoLp-III) is an exchangeable insect
apolipoprotein.
Its function, as currently understood, lies in the stabilization of
low-density
lipophorin particles (LDLp) crossing the hemocoel in phases of high
energy
consumption to deliver lipids from the fat body to the flight muscle
cells.
Recent studies with native Galleria mellonella-apoLp-III gave first
indications
of an unexpected role of that protein in insect immune activation. Here
we report the immune activation by the recombinant protein, documenting
a newly discovered correlation between lipid physiology and immune
defense
in insects. The complete cDNA sequence of G. mellonella-apoLp-III was
identified
by mixed oligonucleotide-primed amplification of cDNA (MOPAC),
3'-RACE-PCR,
and cRACE-PCR. The sequence coding for the native protein was ligated
into
a pET-vector; this construct was transfected into Escherichia coli and
overexpressed in the bacteria. Photometric turbidity assays with human
low density lipoprotein (LDL) and transmission electron microscopy
studies
on apoLp-III-stabilized lipid discs revealed the full functionality of
the isolated recombinant apoLp-III with regard to its lipid-association
ability. For proving its immune-stimulating capacity, apoLp-III was
injected
into the hemocoel of last instar G. mellonella larvae and the
antibacterial
activity in cell-free hemolymph was determined 24 h later. As a result,
the hemolymph samples of injected insects contained strongly increased
antibacterial activities against E. coli as well as clearly enhanced
lysozyme-like
activities. From Northern blot analysis of total RNA from insects
injected
with apoLp-III or the bacterial immune provocator lipopolysaccharide,
it
could be concluded that the transcription rate of apoLp-III mRNA does
not
vary in comparison to untreated last instar larvae.
Insect Biochem. and Mol. Biol. 1999, 29: 989-997.
Institute of Parasitology of the Czech Academy of Sciences, Ceske Budejovice, Czech Republic.
The gut of the adult soft ticks Ornithodoros moubata
displays
high lytic activity against the bacteria Micrococcus luteus.
The
activity differed in the range of two orders of magnitude among
individual
animals and increased in average 4 times during the first week upon the
food uptake. In homogenates of first instar nymphae, the activity
is much lower, nevertheless it grew exponentially towards the molting
day
of the nymphs. The molecule responsible for this activity was
purified
out of the gut content of adult ticks by means of affinity adsorption
on
magnetic-chitin followed by two chromatography steps on cation exchange
FPLC column MonoS.
The homogeneous active protein had a mass of 14 006 ±
20 Daltons as determined by MALDI-TOF mass spectrometry. The
N-terminal
amino-acid sequence of this was K-V-Y-D-R-C-S-L-A-S-E-L-R and had
the best match to the lysozyme from liver of the rainbow trout and
to
lysozymes from digestive tract of several mammals. In addition to it,
the
motif DRCSLA had been known before to be specific for
the digestive lysozymes of Dipteran insect. Taking together, we have
identified
the protein as the tick gut lysozyme. The tick gut lysozyme had high pI
of about 9.7 and retained its full activity after treatment at 60°C
for 30 minutes. The pH optimum of the tick lysozyme was in the range
from
pH 5-7. Only marginal activity could be detected at pH > 8
which
implicated the question about the function of lysozyme in
anti-bacterial
defense in the environment of the tick gut.
FEBS Lett 1999 Sep 3;457(3):522-4
Institut fur Zellbiochemie und klinische Neurobiologie, Universitat Hamburg, Martinistrasse 52, 20246, Hamburg, Germany.
To identify possible ligands of the orphan somatostatin-like
receptor
1 (SLC-1), rat brain extracts were analyzed by using the functional
expression
system of Xenopus oocytes injected with cRNAs encoding SLC-1 and G
protein-gated
inwardly rectifying potassium channels (GIRK). A strong inward current
was observed with crude rat brain extracts which upon further
purification
by cation exchange chromatography and high performance liquid
chromatography
(HPLC) yielded two peptides with a high agonist activity. Mass
spectrometry
and partial peptide sequencing revealed that one peptide is identical
with
the neuropeptide melanin concentrating hormone (MCH), the other
represents
a truncated version of MCH lacking the three N-terminal amino acid
residues.
Xenopus oocytes expressing the MCH receptor responded to nM
concentrations
of synthetic MCH not only by the activation of GIRK-mediated currents
but
also by the induction of Ca(2+) dependent chloride currents mediated by
phospholipase C. This indicates that the MCH receptor can couple either
to the G(i)- or G(q)-mediated signal
transduction pathway, suggesting that MCH may serve for a number of
distinct brain functions including food uptake behavior.
EMBO J. 1999 Nov 1;18(21): 5892-5900
Institut fur Zellbiochemie und klinische Neurobiologie, Universitat Hamburg, Martinistrasse 52, 20246, Hamburg, Germany.
By using degenerate oligonucleotide primers deduced from the
conserved
regions of the mammalian somatostatin receptors, a novel
G-protein-coupled
receptor from Drosophila melanogaster has been isolated
exhibiting
structural similarities to the mammalian somatostain/galanin/opioid
receptors.
To identify the bioactive ligand, a "reberse physiology" strategy was
used
whereby orphan Drosophila receptor-expressing frog oocytes were
screened
against potential ligands. Agonistic activity was
electrophysiologically
recorded as inward potassium currents mediated through co-expressed
G-protein-gated
inwardly rectifying potassium channels (GIRK). Using this approach a
novel
peptide was purified from Drosophila head extracts. Mass spectrometry
revealed
an octapeptide of 925 Da with a sequence SRPYSFGL-NH2 reminiscent of
insect
allatostatin peptides known to control diverse functions such as
juvenile
hormone synthesis during metamorphosis or myogenic visceral muscle
contractions.
Picomolar concentrations of the synthesized octapeptide activated the
cognate
receptor response mediated through GIRK1, indicating that we have
isolated
the 394-amino-acid Drosophila allatostatin receptor which is coupled to
the Gi/Go class of G proteins.
J Biol Chem 1999 Oct 22;274(43):30501-30509
Max von Pettenkofer-Institut, Ludwig Maximilians Universitat, Pettenkoferstrasse 9a, 80336 Munchen, Germany.
RhoGTPases are key regulators of eukaryotic cell physiology. The
bacterial
enteropathogen Salmonella typhimurium modulates host cell physiology by
translocating specific toxins into the cytoplasm of host cells that
induce
responses such as apoptotic cell death in macrophages, the production
of
proinflammatory cytokines, the rearrangement of the host cell actin
cytoskeleton
(membrane ruffling), and bacterial entry into host cells. One of the
translocated
toxins is SopE, which has been shown to bind to RhoGTPases of the host
cell and to activate RhoGTPase signaling. SopE is sufficient to induce
profuse membrane ruffling in Cos cells and to facilitate efficient
bacterial
internalization. We show here that SopE belongs to a novel class of
bacterial
toxins that modulate RhoGTPase function by transient interaction.
Surface
plasmon resonance measurements revealed that the kinetics of formation
and dissociation of the SopE.CDC42 complex are in the same order of
magnitude
as those described for complex formation of GTPases of the Ras
superfamily
with their cognate guanine nucleotide exchange factors (GEFs). In the
presence
of excess GDP, dissociation of the SopE.CDC42 complex was accelerated
more
than 1000-fold. SopE-mediated guanine nucleotide exchange was very
efficient
(e.g. exchange rates almost 10(5)-fold above the level of the
uncatalyzed
reaction; substrate affinity), and the kinetic constants were similar
to
those described for guanine nucleotide exchange mediated by CDC25 or
RCC1.
Far-UV CD spectroscopy revealed that SopE has a high content of
alpha-helical
structure, a feature also found in Dbl homology domains, Sec7-like
domains,
and the Ras-GEF domain of Sos. Despite the lack of any obvious sequence
similarity, our data suggest that SopE may closely mimic eukaryotic
GEFs.
Eur J Biochem 2000 Jan 15;267(2):465-475
Characterization of an alpha-macroglobulin-like glycoprotein isolated from the plasma of the soft tick Ornithodoros moubata
Kopacek, P., Weise, C., Saravanan, T., Vitova, K. and Grubhoffer, L.
Institute of Parasitology of the Czech Academy of Sciences, Ceske Budejovice, Czech Republic.
We report the identification of the first representative of the
alpha-2-macroglobulin
family identified in terrestrial invertebrates. An abundant acidic
glycoprotein
was isolated from the plasma of the soft tick Ornithodoros moubata. Its
molecular mass is about 420 kDa in the native state, whereas in
SDS/PAGE
it migrates as one band of 190 kDa under non-reducing conditions and a
band of 92 kDa when reduced. Chemical deglycosylation reveals that it
is
composed of two different subunits, designated A and B. The N-terminal
amino-acid sequence of subunit A is similar to the N-terminus of
invertebrate
alpha-2-macroglobulin. Sequence analysis of several internal peptides
confirms
that the tick protein belongs to the alpha-2-macroglobulin family, and
the protein is therefore referred to as tick alpha-macroglobulin (TAM).
Functional analyses strengthen this assignment. TAM inhibits trypsin
and
thermolysin cleavage of the high-molecular-weight substrate azocoll in
a manner similar to that of bovine alpha-2-macroglobulin. This effect
is
abolished by pre-treatment of TAM with methylamine. In the presence of
TAM, trypsin is protected against active-site inhibition by soybean
trypsin
inhibitor. We cloned and sequenced a PCR product containing sequences
from
both subunits and spanning the N-terminus of subunit B and the putative
'bait region' (a segment of alpha-2-macroglobulin which serves as
target
for various proteases). This indicates that the two subunits are
generated from a precursor polypeptide by post-translational
processing.
J Biol Chem. 2000 Nov 3;275(44):34359-64.
CBP/p300 activates MyoD by acetylation
Polesskaya, A., Duquet, A., Naguibneva, I., Weise, C., Vervisch, A., Bengal, E., Hucho, F., Robin, P. and Harel-Bellan, A.
Laboratoire Oncogenese, Differenciation et Transduction du Signal, CNRS UPR 9079, Institut Federatif Andre Lwoff, 7 rue Guy Moquet, Villejuif, France.
The myogenic protein MyoD requires two nuclear histone
acetyl-transferases
(HATs), CBP/p300 and PCAF, to transactivate muscle promoters. MyoD is
acetylated
by PCAF in vitro, which seems to increase its affinity for DNA. We here
show that MyoD is constitutively acetylated in muscle cells. In vitro,
MyoD is acetylated both by CBP/p300 and by PCAF on two lysines located
at the boundary of the DNA binding domain. MyoD acetylation by CBP/p300
(as well as by PCAF) increases its activity on a muscle specific
promoter,
as assessed by microinjection experiments. MyoD mutants that cannot be
acetylated in vitro are not activated in the functional assay. Our
results
provide direct evidence that MyoD acetylation functionally activates
the
protein and show that both PCAF and CBP/p300 are candidate enzymes for
MyoD acetylation in vivo.
Bioorganitscheskaja Kimiya 2000; 26, 803-807
MALDI-mass spectrometry used for the identification of novel polypeptides from snake venom
Kukhtina, V.V., Weise, C., Osipow, A.W., Starkov, V.G., Titov, M.I., Esipov, S.E., Ovchinnikova, T.V., Tsetlin, V.I. and Utkin, Yu.N.
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation.
MALDI mass spectrometry, combining high resolution and sensitivity,
can be used to detect and identify the minor components in complex
protein
mixtures. Using this method we searched for new polypeptide compounds,
contained in cobra venoms in low quantities. The isolation of the
compounds
with definite molecular masses by high-performance liquid
chromatography
and subsequent determination of amino-terminal sequence by Edman
degradation
allowed us to identify a number of new proteins in cobra venom woth
molecular
masses in the range of 7-25 kDa, which is typical for the known snake
toxins,
the content of one polypeptide being less than 0.02 % of total protein
content.
Eur J Biochem 2000 Dec 1;267(23):6784-6789.
Muscarinic toxin-like proteins from cobra venom
Kukhtina VV, Weise C, Muranova TA, Starkov VG, Franke P, Hucho F, Wnendt S, Gillen C, Tsetlin VI, Utkin YN
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation.
Three new polypeptides were isolated from the venom of the Thailand
cobra Naja kaouthia and their amino-acid sequences determined. They
consist
of 65-amino-acid residues and have four disulfide bridges. A comparison
of the amino-acid sequences of the new polypeptides with those of snake
toxins shows that two of them (MTLP-1 and MTLP-2) share a high degree
of
similarity (55-74% sequence identity) with muscarinic toxins from the
mamba.
The third polypeptide (MTLP-3) is similar to muscarinic toxins with
respect
to the position of cysteine residues and the size of the
disulfide-confined
loops, but shows less similarity to these toxins (30-34% sequence
identity).
It is almost identical with a neurotoxin-like protein from Bungarus
multicinctus
(TrEMBL accession number Q9W727), the sequence of which has been
deduced
from cloned cDNA only. The binding affinities of the isolated
muscarinic
toxin-like proteins towards the different muscarinic acetylcholine
receptor
(mAChR) subtypes (m1-m5) was determined in competition experiments with
N-[3H]methylscopolamine using membrane preparations from CHO-K1 cells,
which express these receptors. We found that MTLP-1 competed weakly
with
radioactive ligand for binding to all mAChR subtypes. The most
pronounced
effect was observed for the m3 subtype; here an IC50 value of about 3
muM
was determined. MTLP-2 had no effect on ligand binding to any of the
mAChR
subtypes at concentrations up to 1 muM. MTLP-1 showed no inhibitory
effect
on alpha-cobratoxin binding to the nicotinic acetylcholine receptor
from
Torpedo californica at concentrations up to 20 muM.
Toxicon. 2001 Jul 1;39(7):921-927.
First tryptophan-containing weak neurotoxin from cobra venom
Utkin Y.N., Kukhtina V.V., Maslennikov I.V., Eletsky A.V, Starkov V.G., Weise C., Franke P., Hucho F. and Tsetlin V.I.
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation.
The weak neurotoxins from Elapidae venom belong to the group of
"three-finger
toxins" and contain an extra disulfide bridge in the first loop.
Contrary
to other types of "three-finger toxins", there are no X-ray or NMR
structures
for weak neurotoxins, and their pharmacology is largely unknown. With
the
purpose of studying structure-function relationships, we have isolated
a weak neurotoxin (WTX) from the venom of cobra Naja kaouthia using a
combination
of gel-filtration and ion-exchange chromatography. The amino acid
sequence
of the isolated toxin was determined by means of Edman degradation and
MALDI mass spectrometry, the primary structure obtained being confirmed
by 1H-NMR in the course of spatial structure analysis. The WTX sequence
differs slightly from that of the toxin CM-9a isolated earlier from the
same venom (Joubert and Taljaard, 1980). The differences include an
extra
residue (Trp36) between Ser35 and Arg37 as well as interchanging of two
residues (Tyr52 and Lys50) in the C-terminal part of the toxin
molecule.
These changes improve the alignment that can be made with other weak
neurotoxin
sequences. An extended sequence comparison reveals that WTX is the
first
case of a tryptophan-containing weak neurotoxin isolated from cobra
venom.
J Biol Chem. 2001 Mar 2;276(9):6151-6160.
Location of the Polyamine Binding Site in the Vestibule of the Nicotinic Acetylcholine Receptor Ion Channel
Bixel, M.G., Weise, C., Bolognesi, M.L., Rosini, M., Brierly, M., Mellor, I.R., Usherwood, P.N.R., Melchiorre, C. and Hucho, F.
To map the structure of a ligand-gated ion channel we used the
photolabile
polyamine-containing toxin MR44 as photoaffinity label. MR44 binds with
high affinity to the nicotinic acetylcholine receptor in its closed
channel
conformation. The binding stoichiometry was two molecules of MR44 per
receptor
monomer. Upon UV irradiation of the receptor-ligand complex, 125I-MR44
was incorporated into the receptor alpha-subunit. From proteolytic
mapping
studies we conclude that the site of 125I-MR44 cross-linking is
contained
in the sequence alphaHis-186 to alphaLeu-199, which is part of the
extracellular
domain of the receptor. This sequence partially overlaps in its
C-terminal
region with one of the three loops that form the agonist-binding site.
The agonist carbachol and the competitive antagonist
alpha-bungarotoxin
had only minor influence on the photocross-linking of 125I-MR44. The
site
where the hydrophobic head group of 125I-MR44 binds must therefore be
located
outside the zone that is sterically influenced by agonist bound at the
nAChR. In binding and photocross-linking experiments the luminal
non-competitive
inhibitors ethidium and TPMP+ were found to compete with 125I-MR44. We
conclude that the polyamine moiety of 125I-MR44 interacts with the high
affinity non-competitive inhibitor site deep in the channel of the
nAChR,
while the aromatic ring of this compound binds in the upper part of the
ion channel, i.e. in the vestibule, to a hydrophobic region on the
alpha-subunit
that is located in close proximity to the agonist binding site. The
region
of the alpha-subunit labeled by 125I-MR44 should therefore be
accessible
from the luminal side of the vestibule.
FEBS Lett. 2001 Mar 9;492(1-2):95-100.
Characterization of recombinant human nicotinamide mononucleotide adenylyl transferase (NMNAT), a nuclear enzyme essential for NAD synthesis
Schweiger, M., Hennig, K., Lerner, F., Niere, M., Hirsch-Kauffmann, M., Specht, T., Weise, C., Oei, S.L. and Ziegler, M.
Nicotinamide mononucleotide adenylyl transferase (NMNAT) is an essential enzyme in all organisms, because it catalyzes a key step of NAD synthesis. However, little is known about the structure and regulation of this enzyme. In this study we established the primary structure of human NMNAT. The human sequence represents the first report of the primary structure of this enzyme for an organism higher than yeast. The enzyme was purified from human placenta and internal peptide sequences determined. Analysis of human DNA sequence data then permitted the cloning of a cDNA encoding this enzyme. Recombinant NMNAT exhibited catalytic properties similar to the originally purified enzyme. Human NMNAT (molecular weight 31,932) consists of 279 amino acids and exhibits substantial structural differences to the enzymes from lower organisms. A putative nuclear localization signal was confirmed by immunofluorescence studies. NMNAT strongly inhibited recombinant human poly(ADP-ribose) polymerase 1, however, NMNAT was not modified by poly(ADP-ribose). NMNAT appears to be a substrate of nuclear kinases and contains at least three potential phosphorylation sites. Endogenous and recombinant NMNAT were phosphorylated in nuclear extracts in the presence of [-32P]ATP. We propose that NMNAT's activity or interaction with nuclear proteins are likely to be modulated by phosphorylation.
Farmaco 2001 Jan-Feb;56(1-2):133-5
Binding of polyamine-containing toxins in the vestibule of the nicotinic acetylcholine receptor ion channel
Bixel, M.G., Krauss, M., Weise, C., Bolognesi, M.L., Rosini, M., Usherwood, P.N., Melchiorre, C. and Hucho F.
Institut für Biochemie, Freie Universität Berlin, Germany.
Several wasp venoms contain philanthotoxins (PhTXs) that act as noncompetitive inhibitors (NCIs) on cation-selective ion channels including the nicotinic acetylcholine receptor (nAChR). In the search for a ligand with high affinity and specificity for the nAChR we tested a series of newly developed PhTX analogues. Modulation of the structural elements of PhTXs can significantly influence their binding affinities. This approach resulted in the development of the photolabile compound MR44. In photoaffinity labelling studies 125I-MR44 was used to map the ligand-binding site at the Torpedo californica nAChR. Upon UV irradiation of the receptor-ligand complex, 125I-MR44 was mainly incorporated into the receptor alpha-subunit. Proteolytic mapping and microsequencing identified the site of 125I-MR44 cross-linking within the sequence alphaHis-186 to alphaLeu-199 that in its C-terminal region partially overlaps with the agonist-binding site. Since bound agonists had only minor influence on 125I-MR44 photocrosslinking, the site where the hydrophobic head group of 125I-MR44 binds must be located outside the zone that is sterically influenced by agonists bound at the nAChR. A possible site of interaction of 125I-MR44 would be the N-terminal region of the labelled sequence, in which aromatic amino-acid residues are accumulated. We suggest that the polyamine moiety of 125I-MR44 interacts with the high affinity non-competitive inhibitor site deep in the ion channel, while the aromatic ring of this compound binds in the vestibule of the nAChR to a hydrophobic region on the alpha-subunit that is located close to the agonist binding site.
EMBO J 2001 May 15;20(10):2404-12
Regulation of glutamate dehydrogenase by reversible ADP-ribosylation in mitochondria
Herrero-Yraola, A., Bakhit, S.M., Franke, P., Weise, C., Schweiger, M., Jorcke, D. and Ziegler, M.
Institut für Biochemie, Freie Universität Berlin, Thielallee 63, D-14195 Berlin, Germany.
Mitochondrial ADP-ribosylation leads to modification of two proteins
of approximately 26 and 53 kDA: The nature of these proteins and,
hence,
the physiological consequences of their modification have remained
unknown.
Here, a 55 kDa protein, glutamate dehydrogenase (GDH), was established
as a specific acceptor for enzymatic, cysteine-specific
ADP-ribosylation
in mitochondria. The modified protein was isolated from the
mitochondrial
preparation and identified as GDH by N-terminal sequencing and mass
spectrometric
analyses of tryptic digests. Incubation of human hepatoma cells with
[(14)C]adenine
demonstrated the occurrence of the modification in vivo. Purified GDH
was
ADP-ribosylated in a cysteine residue in the presence of the
mitochondrial
activity that transferred the ADP-ribose from NAD(+) onto the acceptor
site. ADP- ribosylation of GDH led to substantial inhibition of its
catalytic
activity. The stoichiometry between incorporated ADP-ribose and GDH
subunits
suggests that modification of one subunit per catalytically active
homohexamer
causes the inactivation of the enzyme. Isolated, ADP-ribosylated GDH
was
reactivated by an Mg(2+)-dependent mitochondrial ADP-ribosylcysteine
hydrolase.
GDH, a highly regulated enzyme, is the first mitochondrial protein
identified
whose activity may be modulated by ADP-ribosylation.
Russian J. Bioorg. Chem. 2001, 27(3), 198-199
Russian version: Bioorg. Khim. 27(3), 224-226 (2001)
Cobra Venom Contains a Protein of the CRISP Family
Osipov, A.V., Weise, C., Franke, P., Kukhtina, V.V., Frings, S., Hucho, F., Tsetlin, V.I. and Utkin, Yu.N.
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation.
Amino-acid sequences of several fragments of the 25k protein
(molecular
mass 24 953 Da) previously isolated from cobra Naja kaouthia (Kukhtina
et al., Bioorg. Khim. 2000, 26, 803-807) were determined. Their
comparison
with the primary structures of known proteins showed that the 25k
protein
belongs to the CRISP family and is the first protein of this type
identified
in cobra venoms.
Angew. Chem. Int. Ed. 40, 3100-3116 (2001); German version: Angew. Chem. 113, 3194-3211 (2001)
Ligand-Gated Ion Channels (Review)
Hucho, F. and Weise, C.
Freie Universität Berlin, Institut für Chemie-Biochemie, Berlin, Germany
Ion channels are at the heart of many biological processes such as
nerve
activity and muscle contraction. How is their impressive ion
selectivity
brought about, how their highly specialised gating? In recent years,
X-ray
crystallography and high-resolution electron microscopy, together with
photo-affinity labeling and mutagenesis techniques in combination with
patch-clamp electrophysiology, have provided a detailed picture of some
channel proteins. Here we summarise the main structural and functional
features based on the advances made mainly within the last decade. We
integrate
these novel insights into a comprehensive description of the class of
ligand-gated
ion channels.
J. Biol. Chem. 2001 Aug 16 [epub ahead of print]
Synaptic Scaffolding Proteins in Rat Brain: Ankyrin Repeats of the Multidomain Shank Protein Family Interact with the Cytoskeletal Protein alpha-Fodrin
Böckers, T.M., Mameza, M.G., Kreutz, M.R., Bockmann, J., Weise, C., Buck, F., Richter, D., Gundelfinger, E.D. and Kreienkamp, H.J.
Institut fuer Zellbiochemie und klinische Neurobiologie, Universitaet Hamburg, Hamburg, Hamburg 20246.
The postsynaptic density is the ultrastructural entity containing
the
neurotransmitter reception apparatus of excitatory synapses in the
brain.
A recently identified family of multidomain proteins termed Shank -
also
known as ProSAP/SSTRIP proteins - plays a central role in organizing
the
subsynaptic scaffold by interacting with several synaptic proteins
including
the glutamate receptors. We used the N-terminal ankyrin repeats of
Shank1
and 3 to search for interacting proteins by yeast-two-hybrid screening
and by affinity chromatography. By cDNA sequencing and mass
spectrometry
the cytoskeletal protein alpha-fodrin was identified as interacting
molecule.
The interaction was verified by pulldown assays and by
coimmunoprecipitation
experiments from transfected cells and brain extracts. Mapping of the
interacting
domains of alpha-fodrin revealed that the highly conserved spectrin
repeat
21 is sufficient to bind to the ankyrin repeats. Both interacting
partners
are coexpressed widely in the rat brain and are colocalized in synapses
of hippocampal cultures. Our data indicate that the Shank1 and 3 family
members provide multiple independent connections between synaptic
glutamate
receptor complexes and the cytoskeleton.
J Bacteriol 2001 Sep;183(18):5343-51
The Brucella suis Homologue of the Agrobacterium tumefaciens Chromosomal Virulence Operon chvE Is Essential for Sugar Utilization but Not for Survival in Macrophages
Alvarez-Martinez, M.-T., Machold, J., Weise, C., Schmidt-Eisenlohr, H., Baron, C. and Rouot, B.
INSERM U431, Universite de Montpellier 2, 34095 Montpellier Cedex 05, France.
Brucella strains possess an operon encoding type IV secretion
machinery
very similar to that coded by the Agrobacterium tumefaciens virB
operon.
Here we describe cloning of the Brucella suis homologue of the
chvE-gguA-gguB
operon of A. tumefaciens and characterize the sugar binding protein
ChvE
(78% identity), which in A. tumefaciens is involved in virulence gene
expression.
B. suis chvE is upstream of the putative sugar transporter-encoding
genes
gguA and gguB, also present in A. tumefaciens, but not adjacent to that
of a LysR-type transcription regulator. Although results of Southern
hybridization
experiments suggested that the gene is present in all Brucella strains,
the ChvE protein was detected only in B. suis and Brucella canis with
A.
tumefaciens ChvE-specific antisera, suggesting that chvE genes are
differently
expressed in different Brucella species. Analysis of cell growth of B.
suis and of its chvE or gguA mutants in different media revealed that
ChvE
exhibited a sugar specificity similar to that of its A. tumefaciens
homologue
and that both ChvE and GguA were necessary for utilization of these
sugars.
Murine or human macrophage infections with B. suis chvE and gguA
mutants
resulted in multiplication similar to that of the wild-type strain,
suggesting
that virB expression was unaffected. These data indicate that the ChvE
and GguA homologous proteins of B. suis are essential for the
utilization
of certain sugars but are not necessary for survival and replication
inside
macrophages.
Cell Tissue Res 2001 Dec;306(3):449-458
Lipophorin of lower density is formed during immune responses in the lepidopteran insect Galleria mellonella.
Dettloff, M., Wittwer, D., Weise, C. and Wiesner A.
Institute of Biology/Zoology, Free University of Berlin, Konigin-Luise-Strasse 1-3, 14195 Berlin, Germany.
Injection of heat-killed bacteria into larvae of the greater wax
moth
Galleria mellonella is followed by changes in lipoprotein composition
in
the hemolymph. Density gradient centrifugation experiments revealed
that
within the first four hours after injection, a part of larval
lipoprotein,
high-density lipophorin (HDLp), was converted into a lipoprotein of
lower
density. SDS-polyacrylamide gel electrophoresis analysis of the
gradient
fractions and sequencing of protein fragments, established that the
exchangeable
apolipoprotein apolipophorin III (apoLp-III), a potent
immune-activator,
was associated with this newly formed lipophorin. To investigate
further
the influence of lipophorin-associated apoLp-III on immune-related
reactions,
we performed in vitro studies with isolated hemocytes from G.
mellonella
and lipophorins from the sphinx moth Manduca sexta, as a natural source
of high amounts of low-density lipophorin (LDLp) and HDLp. The
hemocytes
were activated to form superoxide radicals upon incubation with LDLp,
but
not with HDLp.Fluorescence-labeled LDLp was specifically taken up by
granular
cells. This process was inhibited by adding an excess of unlabeled
LDLp,
but not by HDLp. We hypothesize that larval lipophorin formed in vivo
is
an endogenous signal for immune activation, specifically mediated by
the
binding of lipid-associated apoLp-III to hemocyte membrane receptors.
Biological Chemistry 2002, 383: 1011-1020.
Processing of the Human Transferrin Receptor at Distinct Positions within the Stalk Region by Neutrophil Elastase and Cathepsin G
Kaup, M., Dassler, K., Reineke, U., Weise, C., Tauber, R. and Fuchs, H.
The transferrin receptor (TfR) is a class II transmembrane
glycoprotein
mediating the cellular uptake of iron. The extracellular domain
can
be shed by an unknown membrane-associated protease and appears as
soluble
TfR (sTfR) in blood. The major cleavage site is C-terminal of
Arg-100
within a stalk region that keeps the large extracellular domain at a
distance
of 2.9 nm from the plasma membrane. In many diseases, the sTfR
concentration
is changed and can be utilized for diagnosis and control of
therapy.
However the function of the sTfR remains unclear. In the present
study, we therefore looked for membrane-associated proteases that
cleave
the TfR within the stalk. In plasma membrane fractions of U937
histiocytic
cells we detected two proteolytic activities that cleaved purified TfR
in an in vitro assay producing fragments of either 80 or 85 kDa.
N-terminal sequencing revealed cleavage within the stalk at Val-108 and
Lys-95. Both activities could be inhibited by serine protease
inhibitors
but not by inhibitors of any other class of proteases. Protein
purification
yielded a 28 kDa-protein that could be labeled by the serine protease
inhibitor
[3H]DFP. We showed that this purified protein generated the 80
kDa-fragment.
We examined the substrate specificity of the purified protease by using
a novel protease assay allowing the quantitation of specific
proteolytic
activities on synthetic peptides. This mutational analyses of the
recognition site, the inhibitor profile and the size of the protease
suggested
neutrophil elastase as the unknown protease, which could be proved by
specific
antibodies. Furthermore, purchased neutrophil elastase cleaved
TfR
in our digestion assay at the observed cleavage site at Val-108 whereas
purchased cathepsin G cleaved at Lys-95. In summary, our results
revealed that neutrophil elastase and cathepsin G are present in a
membrane-associated
form in U937 cells and are involved in alternative TfR shedding.
J Cell Biochem 2002;86(2):394-402
Identification of PSF as a protein kinase Calpha-binding protein in the cell nucleus.
Rosenberger U, Lehmann I, Weise C, Franke P, Hucho F, Buchner K.
Freie Universitat Berlin, Institut fur Chemie-Biochemie, AG Neurochemie, Thielallee 63, 14195 Berlin, Germany.
Protein kinase C (PKC) isoforms are present in the cell nucleus in
diverse
cell lines and tissues. Since little is known about proteins
interacting
with PKC inside the cell nucleus, we used Neuro-2a neuroblastoma cells,
in which PKCalpha is present in the nucleus, to screen for nuclear
binding
partners for PKC. Applying overlay assays, we detected several nuclear
proteins which bind to PKCalpha. Specificity of binding was shown by
its
dependence on PKC activation by phorbol ester, calcium, and
phosphatidylserine.
The PKC-binding proteins were partially purified and analyzed by
microsequencing
and mass spectrometry. Four proteins could be identified:
PTB-associated
splicing factor (PSF), p68 RNA helicase, and the heterogeneous nuclear
ribonucleoprotein (hnRNP) proteins A3 and L. In the case of PSF,
binding
to PKC could also be demonstrated in a GST-pull-down assay using
GST-PKCalpha,
expressed in insect cells. Phosphorylation experiments revealed that
PSF
is a weak in vitro substrate for PKCalpha. J. Cell. Biochem. 86:
394-402,
2002. Copyright 2002 Wiley-Liss, Inc.
J Biol Chem. 2002 Oct 11;277(41):38494-502. Epub 2002 Aug 05.
Shedding of the transferrin receptor is mediated constitutively by an integral membrane metalloprotease sensitive to TNFalpha protease inhibitor-2.
Kaup M, Dassler K, Weise C, Fuchs H.
Institut fr Klinische Chemie und Pathobiochemie, Universittsklinikum Benjamin Franklin, Berlin 12200.
The transferrin receptor (TfR) is a transmembrane protein that mediates cellular uptake of iron. Although the serum concentration of the soluble TfR (sTfR) is altered in several diseases and used for diagnostic purposes, the identity and regulation of the shedding protease is unknown. In this study we quantified sTfR-release from microsomal membranes and leukocytic cell lines in the presence of numerous protease inhibitors and cell activating compounds. We show that sTfR-release is mediated by an integral membrane metalloprotease and can be inhibited by inhibitors of matrix metalloproteinases (MMP inhibitor 2) and TNFalpha-shedding (TAPI-2). Cleavage is also inhibited by a specific furin inhibitor indicating that the protease is activated by a furin-like proprotein convertase. Whereas stimulation of the cells by the ectodomain shedding activator phorbol 12-N-myristate 13-acetate (PMA) did not alt sTfR-release significantly, the phosphatase inhibitor pervanadate led to an increase of TfR-shedding in several leukocytic cell lines. Our results suggest that TfR-shedding is constitutively mediated by a member of the metalloprotease family known as ADAM (a disintegrin and metalloprotease).
Insect Biochemistry and Molecular Biology 33 (2003) 103–113
Molecular cloning, expression and isolation of
ferritins
from two
tick species — Ornithodoros moubata and Ixodes
ricinus
Kopacek, P., Zdychová, J. , Yoshiga, T., Weise, C., Rudenko, N. and Law, J.
Institute of Parasitology, Academy of Sciences of the Czech Republic, Ceské Budejovice, Czech Republic
Genes encoding ferritins were isolated and cloned from cDNA libraries of hard tick Ixodes ricinus and soft tick Ornithodoros moubata. Both tick ferritins are composed of 172 amino-acid residues and their calculated mass is 19,667.2 Da and 19,974.5 Da for I. ricinus and O. moubata, respectively. The sequences of both proteins are closely related to each other as well as to the ferritin from another tick species Dermacentor variabilis (>84% similarity). The proteins contain the conserved motifs for ferroxidase center typical for heavy chains of vertebrate ferritins. The stem-loop structure of a putative iron responsive element was found in the 5'- untranslated region of ferritin mRNA of both ticks. Antibodies against fusion ferritin from O. moubata were raised in a rabbit and used to monitor the purification of a small amount of ferritins from both tick species. The authenticity of ferritin purified from O. moubata was confirmed by mass-fingerprinting analysis. In the native state, the tick ferritins are apparently larger (~500 kDa) than horse spleen ferritin (440 kDa). On SDS-PAGE tick ferritins migrate as a single band of about 21 kDa. These results suggest that tick ferritins are homo-oligomers of 24 identical subunits of heavy-chain type. The Northern blot analysis revealed that O. moubata ferritin mRNA level is likely not up-regulated after ingestion of a blood meal.
Insect Biochem Mol Biol. 2003 Aug;33(8):841-51.
Molecular cloning, structure and bait region splice variants of alpha(2)-macroglobulin from the soft tick Ornithodoros moubata.
Saravanan T, Weise C, Sojka D, Kopacek P.
Institute of Parasitology, Academy of Sciences of the Czech Republic and Faculty of Biological Sciences, University of South Bohemia, Branisovska 31, 370 05, Ceske Budejovice, Czech Republic; Institut fur Chemie-Biochemie Freie Universitat Berlin, D-14195 Berlin Germany.
The sequence of a alpha(2)-macroglobulin (alpha(2)M) from the soft tick Ornithodoros moubata (TAM) was determined by cloning and sequencing of overlapping polymerase chain reaction (PCR) and rapid amplification of cDNA ends PCR products. The TAM cDNA sequence is 4944 bp long and contains one open reading frame coding for a protein precursor composed of 1494 amino-acid residues, including a 24-residue signal sequence. The mature protein is cleaved into two subunits similarly to the C3 and C4 components of complement and fish alpha(2)Ms. Phylogeny analysis revealed that TAM is closely related to Limulus alpha(2)M and displays the highest similarity to the partial sequence of alpha(2)M from hard tick Ixodes scapularis. The comparison of conserved cysteine residues between TAM and human and Limulus alpha(2)Ms made it possible to predict the pattern of disulfide bridges and explain the atypical molecular arrangement of TAM. Four variants of the TAM bait region differing only in a short central segment were found; our data indicate that TAM exists as a single-copy gene in the tick genome and its bait region variants likely arise by alternative splicing. TAM is produced by tick hemocytes and it is also significantly expressed in salivary glands. TAM mRNA levels were shown to be up-regulated upon blood meal.
Infect Immun. 2003 Aug;71(8):4326-32.
Characterization of New Members of the Group 3 Outer Membrane Protein Family of Brucella spp.
Salhi I, Boigegrain RA, Machold J, Weise C, Cloeckaert A and Rouot B.
INSERM U431, Universite de Montpellier 2, 34095 Montpellier Cedex 05. Unite BioAgresseurs, Sante et Environnement, INRA, 37380 Nouzilly, France. Institut fur Chemie-Biochemie Freie Universitat Berlin, D-14195 Berlin Germany.
Impairment of the omp25 gene in Brucella spp. leads to attenuated
strains
and confers protection to the host. Omp25 and Omp31, whose functions
remain
unknown, were the first characterized members of group 3 outer membrane
proteins (Omps) (25 to 34 kDa). Recently, genomic and proteomic
approaches
identified five new putative members of this family, some of which are
produced in B. melitensis or B. abortus. In the present study, using
protein
microsequencing, we identified new members of group 3 Omps proteins
produced
in B. suis. Since several monoclonal antibodies (MAbs) against Omp25
cross-reacted
with other members of group 3 Omps, we also performed Western
immunoblotting
to compare wild-type B. suis with mutants systematically having B. suis
omp25-related genes knocked out. We demonstrate the production of three
paralogs of Omp31 and/or Omp25 in B. suis, and the existence of a
common
site of signal peptide cleavage (AXAAD), which is very similar to that
present in the five homologous Omps of Bartonella quintana. The seven
group
3 Omps were classified in four-subgroups on the basis of percentage
amino
acid sequence identities: Omp25 alone, the Omp25b-Omp25c-Omp25d
cluster,
the Omp31/31b subgroup, and the less related Omp22 protein (also called
Omp3b). Together with previous data, our results demonstrate that all
new
members of group 3 Omps are produced in B. suis or in other Brucella
species
and we propose a nomenclature that integrates all of these proteins to
facilitate the understanding of future Brucella interspecies study
results.
Mol Cell. 2003 Nov;12(5):1325-32.
Methylation of histone h3 k4 mediates association of the isw1p ATPase with chromatin.__________________________
Last entry April 28, 2004