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Protein & Peptide Letters
ISSN: 0929-8665
Protein & Peptide Letters
Volume 14, Number 9, 2007
Contents
Review
The Molecular Nature and Consequences of Lipoprotein
(A)’s Association with Platelets Pp. 839-842
D.E. Barre
[Abstract]
Regular Papers
Conservation of Average Hydrophobicity of Apolar Aminoacids
in Polypeptides Constituting Same Glycosyl Hydrolase Sub-Family
Enzymes Pp. 843-845
S. Sengupta
[Abstract]
Biologically Active Peptides Interacting with the
G Protein Coupled Formylpeptide Receptors Pp. 846-853
Y. Le, J.M. Wang, X. Liu, Y. Kong, X. Hou, L. Ruan and
H. Mou
[Abstract]
Freezing Effect on Chirality Generation of DL-Alanine-N-Carboxy
Anhydride Oligomerization in Aqueous Solution Pp.
854-858
T. Vajda and M. Hollósi
[Abstract]
Isolation and Characterization of a Trypsin-Chymotrypsin
Inhibitor from the Seeds of Green Lentil (Lens culinaris)
Pp. 859-864
A.H.K. Cheung and T.B. Ng
[Abstract]
Application of a Chimeric Synthetic Peptide in the
Development of a Serologic Method for the Diagnosis of Hepatitis
G Virus Infection Pp. 865-870
M. Fernández-Vidal, M.D. Cubero, G. Ercilla, M.J.
Gómara and I. Haro
[Abstract]
Digital Coding of Amino Acids Based on Hydrophobic
Index Pp. 871-880
X. Xiao and K.-C. Chou
[Abstract]
Co-Expression and Purification of Recombinant Human
Insulin Like Growth Factor II and Insulin-Like Growth Factor
Binding Protein-6 in Pichia pastoris Yeast
Pp. 876-880
H. Zhou, Z. Chen, H. Chen, S. Li, B. Huang and R. Bi
[Abstract]
Expression, Purification, and Immunological Characterization
of Cr PI Pp. 881-885
H.-Q. Wu, Z.-G. Liu, P.-X. Ran, Z.-W. Zhou and B. Gao
[Abstract]
Inhibition of Sea Urchin Fertilization by Plant Lectins
Pp. 886-893
N.M.R. Macedo, L.V. Costa-Lotufo, C. Pessoa, M.O. Moraes,
L.R. Bonfim and M.V. Ramos
[Abstract]
Protein-Protein and Protein-Ligand Interactions Studied
by Electrospray-Ionization Mass Spectrometry Pp.
894-902
G. Invernizzi, A. Natalello, M. Šamalikova and R.
Grandori
[Abstract]
Robust Quantitative Modeling of Peptide Binding Affinities
for MHC Molecules Using Physical-Chemical Descriptors
Pp. 903-916
O. Ivanciuc and W. Braun
[Abstract]
Design, Synthesis and Biological Evaluation of Antipicornaviral
Pyrrole-Containing Peptidomimetics Pp. 917-922
I. Minchev, S. Vladimirova, L. Vezenkov, A. Bijev, V.
Moussis, L. Nikolaeva-Glomb, V. Tsikaris, M. Czeuz and A.
Galabov
[Abstract]
Crystallization Reports
Crystallization and Preliminary X-Ray Studies of the
Unliganded Wild-Type Bovine Thrombin Pp. 923-924
D. Iyaguchi, M. Yao, N. Watanabe, I. Tanaka and E. Toyota
[Abstract]
Cloning, Expression, Purification, Characterization,
Crystallization and X-Ray Diffraction of Bifunctional Pyrimidine
Deaminase/Reductase from Shigella flexneri 2a
Pp. 925-927
D. Yuan, Q. Wang, W. Gao, F. Sheng, Z. Zhang, Q. Lu, H.
Cang and R. Bi
[Abstract]
Expression, Purification, Crystallization and Preliminary
X-Ray Analysis of Cyan Fluorescent Protein CyPet
Pp. 928-932
Y. Zhou, J. Song, L. Weng, X. Hu, Y. Ding and Z. Zhang
[Abstract]
Abstracts
[Back to top]
The Molecular Nature and Consequences of Lipoprotein
(A)’s Association with Platelets
D.E. Barre
Lipoprotein (a) (Lp (a)) may be pro-thrombotic in humans due
to its apolipoprotein (a) (apo(a))-mediated decreases in fibrinolysis.
Such decreased fibrinolysis arises putatively from interference
with plasminogen conversion to plasmin due to the considerable
homology between apolipoprotein (a) and plasminogen. However,
in vitro, most studies have shown that human Lp (a)
decreases agonist-stimulated platelet aggregation while in
vivo it appears to decrease aggregation as implied by
increased bleeding times with higher blood serum concentrations
of Lp(a). Lp (a) binding to platelets mediated by apo (a)
increases platelet intracellular c-AMP levels in resting platelets,
and decreases platelet pro-duction of thromboxane A2
and fibrinogen binding to platelets all of which reduce platelet
aggregation. One, though not the only, explanation of these
conflicting data may be that Lp(a) self-regulates its interference
with fibrinolysis by reducing platelet aggregation and platelet
binding of fibrinogen and hence the degree of requirement
for fibrinolysis. However, it is concluded more in vivo
work needs to be done to fully understand whether, if at all,
Lp(a) in varying concentrations and isoforms, favours reduced
platelet aggregation or fibrinolysis.
[Back to top]
Conservation of Average Hydrophobicity of Apolar Aminoacids
in Polypeptides Constituting Same Glycosyl Hydrolase Sub-Family
Enzymes
S. Sengupta
Polypeptides constituting the same functional enzyme in cells
of different origins have small sequence similarities among
themselves. Amino acid analysis reveals that each glycosyl
hydrolase sub-family polypeptides conserves an average hydrophobicity
value for total constituent apolar amino acids. The value
may be a measure of the driving force present in the polypeptide
for designed primary collapse for three-dimensional active
site formation.
[Back to top]
Biologically Active Peptides Interacting with the
G Protein Coupled Formylpeptide Receptors
Y. Le, J.M. Wang, X. Liu, Y. Kong, X. Hou, L. Ruan and
H. Mou
Leucocytes accumulate at sites of inflammation and microbial
infection in response to locally produced chemotactic factors.
N-formylpeptides produced by Gram negative bacteria were among
the first chemotactic factors structurally defined which signal
through G protein-coupled formylpeptide receptor (FPR) and
FPR-like 1 (FPRL1) expressed by phagocytic leukocytes in human
and in mouse homogogues mFPR and mFPR2. During the past few
years, a number of pathogen- and host-derived agonists/antagonists
for FPR, FPRL1 and another FPR variant FPR-like 2 (FPRL2)
have been identified. Activation of formylpeptide receptors
(FPRs) in phagocytic leukocytes by agonists results in increased
cell chemotaxis, phagocytosis, and release of pro-inflammatory
mediators. Peptide agonists for FPRs have also been shown
to possess immune adjuvant activity when injected in mice.
In addition, FPR aberrantly expressed on highly malignant
human glioblastoma cells promotes tumor cell migration, proliferation
and production of vascular endothelial growth factor in response
to agonists released by necrotic tumor cells. Therefore, formylpeptide
receptor ligands, by interacting with FPRs, play important
roles in host defense and in the rapid progression of human
glioblastoma.
[Back to top]
Freezing Effect on Chirality Generation of DL-Alanine-N-Carboxy
Anhydride Oligomerization in Aqueous Solution
T. Vajda and M. Hollósi
This article is concerned with a study of the role of ice
in the synthesis of oligopeptides containing L- or D-enantiomeric
excess (ee) from racemic alanine. With this aim, the oligomerization
of DL-alanine-N-carboxyanhydride was investigated by keeping
this activated derivative in liquid (+22oC)
or frozen (-20oC) aqueous
solutions for 30 days. The aqueous solution of the peptide
mixtures were gel-filtered and the aliquots of the fractions
were completely hydrolyzed to alanine monomers. These monomers
were then derivatized with 1-fluoro-2,4-dinitrophenyl-5-L-alanine
amide (Marfey's reagent) and analyzed by RP-HPLC to reveal
the occasional enantiomeric excess of L- or D-Ala. The mass
spectrometry of the gel-filtered fractions pointed to open-chain
peptide mixtures together with a slight amount of cyclic ones,
where the residue numbers ranged between 5-8. Our studies
indicated that an enantiomeric excess of L- or D-Ala appeared
in some oligopeptide fractions. Their excesses were significantly
larger in the frozen than liquid solution. Speculations are
made as concerns the implications of our findings in the events
of prebiotic chemistry.
[Back to top]
Isolation and Characterization of a Trypsin-Chymotrypsin
Inhibitor from the Seeds of Green Lentil (Lens culinaris)
A.H.K. Cheung and T.B. Ng
A Bowman-Birk type trypsin-chymotrypsin inhibitor was isolated
from seeds of the legume green lentil (Lens culinaris)
by means of affinity chromatography on Affi-gel blue gel,
ion exchange chromatography on Q-Sepharose, ion exchange chromatography
by fast protein liquid chromatography (FPLC) on Mono Q and
Mono S, and gel filtration by FPLC on Superdex 75. The trypsin-chymotrypsin
inhibitor was bound on the first three types of chromatographic
media. It appeared as a single 16-kDa peak in gel filtration
and a single 16-kDa band in sodium dodecyl sulfate-polyacrylamide
gel electrophoresis. The trypsin inhibitory activity of the
inhibitor was sensitive to the reducing agent dithiothreitol.
It was completely abrogated after treatment with 10 mM dithiothreitol
for 20 minutes. The protease inhibitor did not exert any inhibitory
effect on hepatoma (Hep G2) and breast cancer (MCF 7) cell
lines. There was no suppressive action on several fungal species
including Botrytis cinerea, Fusarium oxysporum and Mycosphaerella
arachidicola. It slightly inhibited the activity of HIV-1
reverse transcriptase, with an IC50
of 30 mM.
[Back to top]
Application of a Chimeric Synthetic Peptide in the
Development of a Serologic Method for the Diagnosis of Hepatitis
G Virus Infection
M. Fernández-Vidal, M.D. Cubero, G. Ercilla, M.J.
Gómara and I. Haro
New putative antigenic peptides corresponding to the N- and
C-terminal of the E2 envelope protein of GBV-C/HGV were synthesized
using solid-phase chemistry. The antigens were obtained in
linear and chimeric forms with the main aim of improving the
sensitivity of the enzyme immunoassays. Furthermore, CD and
FTIR have been used in conjunction to characterize their conformational
changes showing that the chimeric peptide presents a more
ordered secondary structure than its parent peptides.
[Back to top]
Digital Coding of Amino Acids Based on Hydrophobic
Index
X. Xiao and K.-C. Chou
Analysis of amino acid sequences can provide useful insights
into the tertiary structures of proteins and their biological
functions. One of the critical problems in amino acid analysis
is how to establish a digital coding system to better reflect
the properties of amino acids and their degeneracy. Based
on the hydrophobic index, a one-to-one relationship has been
established between the amino acid sequence and the digital
signal process. Such a “bridge” will make it possible
to apply all the existing powerful methods in the signal processing
area to analysis of the amino acid sequences.
[Back to top]
Co-Expression and Purification of Recombinant Human
Insulin Like Growth Factor II and Insulin-Like Growth Factor
Binding Protein-6 in Pichia pastoris Yeast
H. Zhou, Z. Chen, H. Chen, S. Li, B. Huang and R. Bi
For the preparation of the complex of IGF-II and IGFBP-6,
a co-expression vector containing two copies of human IGF-II
and IGFBP-6 expression cassette was constructed with alcohol
oxidase (AOX1) promoter and secretion signal sequence of α-factor,
and transformed to Pichia pastoris yeast. Through
a purification procedure involving anion-exchange chromatography
and gel filtration, a complex of IGF-II with IGFBP-6 was obtained.
An additional C-terminal sequence of IGFBP-6 (CS-BP6) was
found to be bound to this complex. Dynamic light scattering
showed that this complex was very stable and homogenous in
solution. Western blotting based on non-reducing Tricine-SDS-PAGE
indicated that IGF-II expression coupled with IGFBP-6 might
significantly avoid the mispairing of disulfide bonds compared
with the IGF-II expressed alone.
[Back to top]
Expression, Purification, and Immunological Characterization
of Cr PI
H.-Q. Wu, Z.-G. Liu, P.-X. Ran, Z.-W. Zhou and B. Gao
An efficient preparation of Periplaneta americana
nymphae allergen, Cr PI (54 kDa) is described. It was expressed
as a GST-tag fusion protein in Escherichia coli,
strain BL21 (DE3). Expression of recombinant Cr PI (rCr PI),
de-naturation/renaturation of the inclusion bodies and the
effects of protein and L-arginine concentration on inclusion
body aggregation were optimized. The fusion protein was purified
by affinity chromatography and size exclusion chromatography,
and Cr PI fusion protein was purified to >95%. rCr PI bound
strongly to IgE in the sera of individuals with cockroach
allergies as shown by western blot and ELISA. Highly refolded
and purified recombinant protein was obtained, providing a
basis for the large-scale preparation of Cr PI allergen.
[Back to top]
Inhibition of Sea Urchin Fertilization by Plant Lectins
N.M.R. Macedo, L.V. Costa-Lotufo, C. Pessoa, M.O. Moraes,
L.R. Bonfim and M.V. Ramos
Effects of plant lectins on sea urchin (Lytechinus variegatus)
fertilization and a partial characterization of lectin-binding
involved in the process were evaluated. IC50
doses for inhibition of fertilization varied from 4.1 to 135.5
μg/ml
when the lectins were pre-incubated with sperms and from 0.7
to 33.4 μg/ml
when pre-incubated with eggs. Such effects were reversed when
the lectins were heat inactivated. FITC-labeled lectins bound
egg surfaces while their denatured forms did not. Glucose/mannose
specific lectins bound weaker to eggs when pre-incubated with
the glycoprotein bovine lactotransferrin. None of the glycoproteins
assayed diminished FITC patterns of the Gal/GalNAc binding
lectins. Pre-incubation of Glucose/mannose binding lectins
with eggs did not alter binding of Gal/GalNAc lectins. Lectins
with distinct competencies for binding monosaccharide and
glycoconjugates were able to inhibit sea urchin fertilization.
[Back to top]
Protein-Protein and Protein-Ligand Interactions Studied
by Electrospray-Ionization Mass Spectrometry
G. Invernizzi, A. Natalello, M. Šamalikova and R.
Grandori
Preservation of non-covalent interactions in biopolymer mass
spectrometry offers new approaches to binding analysis. Recent
work from our laboratory is reviewed here and discussed with
reference to recent literature in the field. Three issues
are considered in particular: hydrophobically stabilized complexes,
pH-dependent transitions, and linked protein-ligand and protein-protein
binding equilibria.
[Back to top]
Robust Quantitative Modeling of Peptide Binding Affinities
for MHC Molecules Using Physical-Chemical Descriptors
O. Ivanciuc and W. Braun
Major histocompatibility complex (MHC) molecules bind short
peptides resulting from intracellular processing of foreign
and self proteins, and present them on the cell surface for
recognition by T-cell receptors. We propose a new robust approach
to quantitatively model the binding affinities of MHC molecules
by quantitative structure-activity relationships (QSAR) that
use the physical-chemical amino acid descriptors E1-E5.
These QSAR models are robust, sequence-based, and can be used
as a fast and reliable filter to predict the MHC binding affinity
for large protein databases.
[Back to top]
Design, Synthesis and Biological Evaluation of Antipicornaviral
Pyrrole-Containing Peptidomimetics
I. Minchev, S. Vladimirova, L. Vezenkov, A. Bijev, V.
Moussis, L. Nikolaeva-Glomb, V. Tsikaris, M. Czeuz and A.
Galabov
A series of new peptidomimetics based on the tripeptide sequence
Z-Leu-Phe-Gln-OH were synthesized, with ten of these including
the a-nitrogen atom of the N-terminal amino acid incorporated
into the pyrrole cycle. The synthesized compounds were tested
for antiviral activity by agar-diffusion plaque inhibition
test against Coxsackievirus B1 replication in FL cell. Four
of the products were observed to possess an antiviral activity,
which was proven to be significant for one product. N- terminal
pyrrole moiety and C-terminal free carboxyl function are available
in all active compounds. On the other hand, their corresponding
–OBzl and -OBut esters
are inactive.
[Back to top]
Crystallization and Preliminary X-Ray Studies of the
Unliganded Wild-Type Bovine Thrombin
D. Iyaguchi, M. Yao, N. Watanabe, I. Tanaka and E. Toyota
Wild type of bovine thrombin has been crystallized in a ligand-free
form by the hanging drop vapor diffusion method with polyethylene
glycol 4000 and 2-propanol. The crystals belong to space group
P433212
with unit cell parameters of a = b = 87.7 Å, c = 195.9
Å. X-ray diffraction data were collected to 2.8 Å
resolution.
[Back to top]
Cloning, Expression, Purification, Characterization,
Crystallization and X-Ray Diffraction of Bifunctional Pyrimidine
Deaminase/Reductase from Shigella flexneri 2a
D. Yuan, Q. Wang, W. Gao, F. Sheng, Z. Zhang, Q. Lu, H.
Cang and R. Bi
Bifunctional pyrimidine deaminase/reductase (RibD) plays an
important role during riboflavin biosynthesis in many microorganisms.
The 40.4 kDa RibD from Shigella flexneri 2a has been
cloned, expressed, purified and characterized. Three Crystals
of RibD have been obtained by the hanging-drop technique at
291 K using PEG 20k or NaCl as pre-cipitant. The RibD crystal
using PEG 20k as precipitant diffracted to 2.5Å.
[Back to top]
Expression, Purification, Crystallization and Preliminary
X-Ray Analysis of Cyan Fluorescent Protein CyPet
Y. Zhou, J. Song, L. Weng, X. Hu, Y. Ding and Z. Zhang
The technique of fluorescence (or Förster) resonance
energy transfer (FRET) is widely used to observe bimolecular
interaction in living cells. Cyan and yellow fluorescent proteins
are the most widely used pair in FRET analysis. CyPet and
YPet are two newly optimized fluorescent proteins that have
much better dynamic range and sensitivity than CFP/YFP pair,
although the crystallographic structure and the mechanism
of better fluorescent characteristics of CyPet are still unknown.
We have expressed the cyan fluorescent protein CyPet using
pT7 prokaryocyte expression system in Escherichia coli
strain Rosetta (DE3) pLysS by auto-induction. After purification,
the recombinant CyPet protein was crystallized by hanging
drop vapor diffusion technique and could diffract to 2.55Å
resolution. The data showed that the orthorhombic CyPet crystal
was in space group P212121 with unit cell parameters (51.55,
61.53, 63.36) and contained one molecule in one asymmetric
unit.
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