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Protein & Peptide Letters
ISSN: 0929-8665
Protein & Peptide Letters
Volume 13, Number 2, 2006
Contents
Regular Papers
Autoantibody Reaction to Myelin Basic Protein
by Plasma Parvovirus B19 IgG in MS Patients Pp. 109-111
G. Thomas, L. Rael, R. Shimonkevitz, I. Melamed and D.
Bar-Or
[Abstract]
Effect of a New Ionic Pair on the
Unfolding Activation Barrier of β-Glucosidase B
Pp. 113-118
R.A. Zubillaga, E. García-Hernández, M. Camarillo-Cadena,
M. León and J. Polaina
[Abstract]
Processing of Amyloid β-Peptides by Neutral Cysteine
Protease Bleomycin Hydrolase Pp. 119-123
A. Kajiya, H. Kaji, T. Isobe and A. Takeda
[Abstract]
A Nonradioactive Assay for Poly(A)-Specific
Ribonuclease Activity by Methylene Blue Colorimetry Pp.
125-128
Y. Cheng, W.-F. Liu, Y.-B. Yan and H.-M. Zhou
[Abstract]
Mitochondria-Regulated Death Pathway Mediates
(DIPP-L-Leu)2-L-LysOCH3-Induced
K562 Cells Apoptosis. Pp. 129-134
J. Yang, Y. Jiang, F. Liu and Y. Zhao
[Abstract]
Synthesis, Characterization and Biological Activity
of Chemically Modified Insulin Derivative with Alpha Lipoic
Acid Pp. 135-142
T. Huang and K. Huang
[Abstract]
Timing of Mutation in Influenza A Virus Hemagglutinins
by Means of Amino-Acid Distribution Rank and Fast Fourier
Transform Pp. 143-148
G. Wu and S. Yan
[Abstract]
Asparaginase Display of Human Cholesteryl Ester
Transfer Protein (CETP) B Cell Epitopes for Inducing High
Titers of Anti-CETP Antibodies In Vivo
Pp. 149-154
Q. Gaofu, C. Rongyue, M. Dan, Z. Xuejun, W. Xuejun, W. Jie
and L. Jingjing
[Abstract]
High-Level Expression of Human β-Defensin-2
Gene with Rare Codons in E. coli Cell-Free System
Pp. 155-161
H. Chen, Z. Xu, N. Xu and P. Cen
[Abstract]
LYS70 of E. coli Quinolinate Phosphori-bosyltransferase
Is Protected from Chemical Modification by Formation of an
Inhibitor Complex. Pp. 163-167
R. Gaur, T. Roberts and K. Calvo
[Abstract]
Influence of Human β-Casomorphin-7
on Specific Binding of 3H-Spiperone to the 5-HT2-Receptors
of Rat Brain Frontal Cortex Pp. 169-170
O.Y. Sokolov, N.V. Kost, Y.A. Zolotarev, E.N.
Ryukert and A.A. Zozulya
[Abstract]
Identification and Characterization of Trichoplusia
ni Furin Truncated Mutant Pp. 171-175
L. Wang, D. Xu, M. Qian, G. Yang and X. Wu
[Abstract]
Structure-Based Stabilization of an Enzyme: The
Case of Penicillin Acylase from Alcaligenes faecalis
Pp. 177-183
T. Wang, H. Zhu, X. Ma, Y. Ma and D. Wei
[Abstract]
Effects of [Nphe1]nociceptin(1-13)NH2,
[Phe1(CH2-NH)Gly2]nociceptin(1-13)NH2,
and Nocistatin on Nociceptin Inhibited Constrictions of Guinea
Pig Isolated Bronchus Pp. 185-188
Q. Yang, Y. Chen, C. Fu, M. Chang, Y. Peng and R. Wang
[Abstract]
Solid Phase Peptide Synthesis in Water
VI: Evaluation of Water-Soluble Coupling Reagents for Solid
Phase Peptide Synthesis in Aqueous Media Pp. 189-192
K. Hojo, M. Maeda, N. Tanakamaru, K. Mochida
and K. Kawasaki
[Abstract]
Eph/ephrin Membrane Proteins: A Mammalian Expression
Vector pTIg-BOS-Fc Allowing Rapid Protein Purification
Pp. 193-196
B.W. Day, F.M. Smith, K. Chen, J.K. McCarron, N.I. Herath,
M. Lackmann and A.W. Boyd
[Abstract]
Expression and Purification of Active WW Domains
of FBP11/HYPA and FBP28/ CA150 Pp. 197-201
Y. Kato, Y. Sawano and M. Tanokura
[Abstract]
Crystallization Reports
Expression, Purification, Crystallization and Preliminary
X-Ray Analysis of Human Spindlin1, an Ovarian Cancer-Related
Protein Pp. 203-205
F. Jiang, Q. Zhao, L. Qin, H. Pang, X. Pei and Z. Rao
[Abstract]
Crystallization and Preliminary Diffraction Studies
of Malate Dehydrogenase from Streptomyces aureofaciens
Pp. 207-210
D. Mikulášová,
N. Tomášková,
J. Maderová and M. Kollárová
[Abstract]
Abstracts
[Back to top]
Autoantibody Reaction to Myelin Basic Protein by Plasma
Parvovirus B19 IgG in MS Patients
G. Thomas, L. Rael, R. Shimonkevitz, I. Melamed and D.
Bar-Or
The etiology of multiple sclerosis (MS) remains unclear.
To determine if autoantibodies to myelin basic protein (MBP)
are produced during parvovirus B19 infection, a competitive
ELISA was performed using plasma from MS patients exhibiting
high IgG titers for parvovirus. Our results showed the addition
of MBP decreased the binding of IgG to B19 antigen in a dose
dependent fashion suggesting a possible link between parvovirus
B19 and a subset of patients with clinical MS.
[Back to top]
Effect of a New Ionic Pair on the Unfolding Activation
Barrier of β-Glucosidase B
R.A. Zubillaga, E. García-Hernández, M. Camarillo-Cadena,
M. León and J. Polaina
Thermal unfolding kinetics of β-glucosidase
B from Paenibacillus polymyxa and its thermoresistant
mutant H62R were determined from far-UV circular dichroism
(CD) measurements at different temperatures. The unfolding
of both enzymes followed simple two-state kinetics. The new
ionic pair formed between Arg62 and Glu429 in the H62R variant
did not change substantially the enzyme structure as judged
by far-UV CD and fluorescence spectra, but produced an increase
in the unfolding activation barrier of 0.95 ± 0.10
kcal mol-1, in good agreement with the energetic
contribution reported for surface salt bridges in proteins.
Eyring’s analysis of the unfolding kinetic constants
showed that the activation enthalpies for thermal denaturation
of both enzymes were essentially the same. Thus, the greater
kinetic stability rendered by the salt bridge seems to be
due to a reduction in the activation entropy.
[Back to top]
Processing of Amyloid β-Peptides by Neutral Cysteine
Protease Bleomycin Hydrolase
A. Kajiya, H. Kaji, T. Isobe and A. Takeda
We studied the processing of amyloid β-peptides
(Aβs)
including Aβ1-40,
Aβ1-42
and pAβ3-42
by rat neutral cysteine protease bleomycin hydrolase (BH)
according to the methods of SDS-PAGE, HPLC and matrix-assisted
laser desorption/inonization time-of-flight mass spectrometry
(MALDI-TOF MS). BH significantly processed them by novel features
of its diverse activities. It initially cleaved at two sites,
His14-Gln15 and Phe19-Phe20
bonds, in Aβ1-40
and Aβ1-42
by endopeptidase activity. The resultant peptides were degraded
to short intermediates then to amino acids by aminopeptidase
and/or carboxypeptidase activities. Also, full-length Aβs
were clipped at the carboxyl(C)-terminal region. On the other
hand, BH cleaved at only the His14-Gln15
bond in pβA3-42
within a short period of the reaction by endopeptidase activity,
and processed the intermediates in order by carboxypeptidase
activity. On processing by BH, it found that both fibrillar
Aβ1-40
and Aβ1-42
were more resistant than non-fibrillar peptides. These results
indicate that the processing specificity of BH depends upon
the structure and sequence of Aβs.
[Back to top]
A Nonradioactive Assay for Poly(A)-Specific Ribonuclease
Activity by Methylene Blue Colorimetry
Y. Cheng, W.-F. Liu, Y.-B. Yan and H.-M. Zhou
A simple nonradioactive assay, which was based on the specific
shift of the absorbance maximum of methylene blue induced
by its intercalation into poly(A) molecules, was developed
for poly(A)-specific ribonuclease (PARN). A good linear relationship
was found between the absorbance at 662 nm and the poly(A)
concentration. The assay conditions, including the concentration
of methylene blue, the incubation temperature and time, and
the poly(A) concentration were evaluated and optimized.
[Back to top]
Mitochondria-Regulated Death Pathway Mediates (DIPP-L-Leu)2-L-LysOCH3-Induced
K562 Cells Apoptosis.
J. Yang, Y. Jiang, F. Liu and Y. Zhao
(DIPP-L-Leu)2-L-LysOCH3
is a diisopropylphosphoryl dipeptide which is known to induce
apoptosis of human leukemia K562 cells. The molecular and
cellular mechanisms involved in this process remain to be
clarified. Herein, we show that (DIPP-L-Leu)2-L-LysOCH3-induced
apoptosis is associated with cytosolic accumulation of cytochrome
c, sustained loss of mitochondrial transmembrane potential
(MMP), transient generation of reactive oxygen species (ROS)
and elevation of intracellular Ca2+ concentration.
A specific caspase assay reveals an increase in caspase-9
and caspase-3 activity but no change in caspase-8 activity.
Immunofluorescence analysis indicates that (DIPP-L-Leu)2-L-LysOCH3
induced upregulation of pro-apoptotic Bax and downregulation
of anti-apoptotic Bcl-2 and Bcl-xL. These results
suggest that the mitochondria-regulated death pathway mediates
(DIPP-L-Leu)2-L-LysOCH3-induced
K562 cells apoptosis.
[Back to top]
Synthesis, Characterization and Biological Activity
of Chemically Modified Insulin Derivative with Alpha Lipoic
Acid
T. Huang and K. Huang
A novel chemically-modified insulin, ε-NB29-lipoyl
insulin, was selectively prepared by the covalent linkage
of α-lipoic
acid (LA) to the ε-amino
group of LysB29 of insulin without any protecting
agent and analyzed by PAGE, HPLC, MALDI-TOF-MS. Monolipoyl-
insulin maintained the glucose-lowering effect as well as
native insulin and showed a longer duration of action than
native insulin and an inhibitory effect towards trypsin degradation.
[Back to top]
Timing of Mutation in Influenza A Virus Hemagglutinins
by Means of Amino-Acid Distribution Rank and Fast Fourier
Transform
G. Wu and S. Yan
In this study, we calculated the amino-acid distribution
rank of 1201 hemagglutinins from influenza A viruses dated
from 1918 to 2004 in order to compare them with respect to
subtypes, species and years. After noticing fluctuations in
distribution rank along the time course, we used the fast
Fourier transform to determine the mutation periodicity of
the hemagglutinins. Then we estimated our position at the
current cycle of hemagglutinin evolutionary process to determine
how many years remain before the next possible outbreak of
influenza and bird flu. Finally, we used the trend channel
to outlook the future of hemagglutinins for the next half
a century. As our study covers almost all the full-length
amino-acid sequences of hemagglutinins from various influenza
A viruses, the conclusions will be valid for years until the
number of hemagglutinins in Protein Databank is significantly
increased.
[Back to top]
Asparaginase Display of Human Cholesteryl Ester Transfer
Protein (CETP) B Cell Epitopes for Inducing High Titers of
Anti-CETP Antibodies In Vivo
Q. Gaofu, C. Rongyue, M. Dan, Z. Xuejun, W. Xuejun, W. Jie
and L. Jingjing
The recombinant chimeric enzyme, AnsB-TTP-CETPC, comprising
asparaginase, tetanus toxin helper T cell epitope and human
CETP B cell epitope was expressed as a soluble protein in
Escherichia coli. The purified chimeric enzyme exhibited
approximate 83% activity of the native asparaginase. After
immunization with three doses of chimeric enzyme, high titers
of anti-CETP antibodies were induced and lasted more than
eighteen weeks in mice, and could even be detected at a dilution
of 1:12800 by normal ELISA assay. The specificity of anti-CETP
antibody was verified by Western blot assay. After displaying
on the surface of asparaginase, the weak antigenicity of CETP
epitope was effectively overcome, there after a strong CETP-specific
immune response was evoked in mice immunized with the chimeric
enzyme. Histochemical analysis of mice kidney tissue showed
that immunization with the chimeric enzyme did not cause any
pathological changes in mice. Collectively, the chimeric enzyme
may be further developed as a vaccine against atherosclerosis
in the future.
[Back to top]
High-Level Expression of Human β-Defensin-2 Gene
with Rare Codons in E. coli Cell-Free System
H. Chen, Z. Xu, N. Xu and P. Cen
Human β-defensin-2
(hBD2) is a small cationic peptide with a broad range of antimicrobial
activity. An E. coli cell-free system was employed
to express the hBD2 fusion protein by using the hBD2 gene
with 14 rare codons. The results showed that the expression
level of trxA-hBD2 fusion protein was 0.35 mg/ml,
which is the same as that obtained with a synthetic codon-optimized
gene. By using another fusion partner (GFP), similar high-level
expression was also achieved in this cell-free system. This
meant that human beta-defensin-2 gene could be directly used
to express hBD2 fusion protein efficiently in an E. coli
cell-free system without the optimization of codons.
The expression level of hBD2 fused with thioredoxin could
be further improved up to 2.0 mg/ml by adopting a continuous
exchange cell-free system. A simple one-stage affinity purification
procedure was also developed to recover this fusion protein
efficiently.
[Back to top]
LYS70 of E. coli Quinolinate Phosphori-bosyltransferase
Is Protected from Chemical Modification by Formation of an
Inhibitor Complex.
R. Gaur, T. Roberts and K. Calvo
Quinolinate phosphoribosyltransferase was examined for susceptibility
to different chemical modification reagents. Loss of enzyme
activity with trinitrobenzenesulfonate (TNBS) occurred when
1.1 lysines per subunit were modified. Tryptic digestion of
the modified enzyme followed by HPLC-MS analysis of the peptides
showed Lys70 reacts with TNBS. Based on x-ray studies, this
amino acid participates in a conformational change distant
from the active site.
[Back to top]
Influence of Human β-Casomorphin-7 on
Specific Binding of 3H-Spiperone to the 5-HT2-Receptors
of Rat Brain Frontal Cortex
O.Y. Sokolov, N.V. Kost, Y.A. Zolotarev, E.N.
Ryukert and A.A. Zozulya
Using radio-receptor analysis, it has been demonstrated that
human β-casomorphin-7
(Tyr-Pro-Phe-Val-Glu-Pro-Ile) displaces 3H-spiperone
from 5-HT2-receptors of rat brain frontal cortex.
IC50 of human β-casomorphin-7
was 8 μM.
These data suggest that one of the meachanisms of neurotropic
action of β-casomorphin-7
is might be associated with its influence on the serotoninergic
system.
[Back to top]
Identification and Characterization of Trichoplusia
ni Furin Truncated Mutant
L. Wang, D. Xu, M. Qian, G. Yang and X. Wu
We report a new prohormone convertase gene of insect origin,
Trichoplusia ni furin, which was cloned from BTI-Tn-5B-4
(Tn5) insect cells. We constructed a truncated mutant that
lacked Cys-rich repeated segments. Using baculovirus expression
system and standard enzymatic assay, we obtained recombinant
Tn furin and evaluated aspects of its function.
[Back to top]
Structure-Based Stabilization of an Enzyme:
The Case of Penicillin Acylase from Alcaligenes faecalis
T. Wang, H. Zhu, X. Ma, Y. Ma and D. Wei
The modeled structure of penicillin acylase from Alcaligenes
faecali (AFPGA) was constructed by comparative modeling
with the Modeller program. Candidate positions that could
be replaced with cysteine were estimated by scanning the modeled
structure of AFPGA with the program MODIP (modeling disulfide
bond in protein). The mutant Q3C/P751C had a higher optimum
temperature by three degrees than that of the wild type AFPGA.
The half life of the double mutant Q3C/P751C at 55°C was
increased by 50%. To our knowledge, this was the first structure-based
genetic modification of AFPGA.
[Back to top]
Effects of [Nphe1]nociceptin(1-13)NH2,
[Phe1(CH2-NH)Gly2]nociceptin(1-13)NH2,
and Nocistatin on Nociceptin Inhibited Constrictions of Guinea
Pig Isolated Bronchus
Q. Yang, Y. Chen, C. Fu, M. Chang, Y. Peng and R. Wang
Electric field stimulation (EFS) causes excitatory non adrenergic-non
cholinergic (eNANC) and cholinergic constrictions in the guinea
pig isolated bronchus, the activation of eNANC and cholinergic
nerves respectively. We investigated the effects of [Nphe1]nociceptin(1-13)NH2
([Nphe1]NC(1-13)NH2), [Phe1(CH2-NH)Gly2]nociceptin(1-13)NH2
([F/G] NC(1-13)NH2), and nocistatin (NST) on nociceptin
(NC) inhibited constrictions in isolated bronchus of guinea
pig. The results show that NC (1 µmol/L) inhibited EFS-induced
eNANC and cholinergic constrictions compared with the control,
in which nociceptin was not applied. After pretreatment with
[Nphe1]NC(1-13)NH2, [F/G]NC(1-13)NH2,
or NST, the inhibitions of NC were antagonized by [Nphe1]NC(1-13)NH2
and [F/G]NC(1-13)NH2 but not NST. However, [Nphe1]NC(1-13)NH2
[F/G]NC(1-13)NH2, and NST did not affect the inhibitions
induced by morphine. Furthermore, [Nphe1]NC(1-13)NH2,
[F/G]NC(1-13)NH2 and NST did not cause any appreciable
effects on EFS-induced eNANC and cholinergic constrictions
in guinea pig bronchi. The results demonstrate that [Nphe1]NC(1-13)NH2
and [F/G]NC(1-13)NH2 but not NST act as selective
antagonists of the NC receptor and the effects of NC on EFS-induced
constrictions of guinea pig isolated bronchus.
[Back to top]
Solid Phase Peptide Synthesis in Water VI:
Evaluation of Water-Soluble Coupling Reagents for Solid Phase
Peptide Synthesis in Aqueous Media
K. Hojo, M. Maeda, N. Tanakamaru, K. Mochida
and K. Kawasaki
Solid phase peptide synthesis requires large amounts of organic
solvents, the safe disposal of which is an important environmental
issue. Peptide synthesis, if performed in water and using
less or nontoxic reagents, circumvents the disposal problem.
Our ultimate aim is to develop an “environment-friendly”
solid phase peptide synthesis (SPPS) methodology. Previously,
we showed that SPPS in water is feasible. To perform SPPS
in water, the coupling reagent must be water-soluble and maintain
its reactivity in water. For this report, we tested the efficacy
of the water-soluble coupling reagents, 2-(5-norbornene-2,3-dicarboximido)-1,1,3,3-tetramethyluronium
tetrafluoroborate (TNTU) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium
chloride (DMT-MM), towards SPPS in water. We successfully
synthesized Leu-enkephalin amide on a solid support suspended
in aqueous 50% EtOH using DMT-MM and 2-(4-sulfophenylsulfonyl)ethoxycarbonylamino
acids.
[Back to top]
Eph/ephrin Membrane Proteins: A Mammalian Expression
Vector pTIg-BOS-Fc Allowing Rapid Protein Purification
B.W. Day, F.M. Smith, K. Chen, J.K. McCarron, N.I. Herath,
M. Lackmann and A.W. Boyd
There is an urgent need for high purity, single chain, fully
functional Eph/ephrin membrane proteins. This report outlines
the pTIg-BOS-Fc vector and purification approach resulting
in rapid increased production of fully functional single chain
extracellular proteins that were isolated with high purity
and used in structure-function analysis and pre-clinical studies.
[Back to top]
Expression and Purification of Active WW Domains of
FBP11/HYPA and FBP28/ CA150
Y. Kato, Y. Sawano and M. Tanokura
Production of GST-fused WW domains of FBP proteins was increased
using the bubbling cultivation method for E. coli.
Purified WW domains of FBP11 and FBP28 bound a PL motif peptide
with dissociation constants (KD) of 248
± 27 and 1880 ± 280 μM,
respectively.
[Back to top]
Expression, Purification, Crystallization and Preliminary
X-Ray Analysis of Human Spindlin1, an Ovarian Cancer-Related
Protein
F. Jiang, Q. Zhao, L. Qin, H. Pang, X. Pei and Z. Rao
Human spindlin1 is a newly screened and identified gene product
related to ovarian carcinomas and is highly homologous to
mouse spindlin. It is an abundant maternal transcript expressed
in the mouse during the transition from oocyte to embryo.
Here, the recombinant human spindlin1 has been overexpressed
in Escherichia coli BL21, purified and crystallized
using the hanging-drop vapour-diffusion method. Crystals diffracting
to 2.25 Å resolution were obtained using ammonium sulfate
as precipitant. The crystals belong to the space group P212121,
with unit-cell parameters a =40.7 Å, b =84.4 Å,
c =136.4 Å, α=β=γ=90°.
Assuming two molecules per asymmetric unit, the solvent content
is calculated to be 42.4%.
[Back to top]
Crystallization and Preliminary Diffraction Studies
of Malate Dehydrogenase from Streptomyces aureofaciens
D. Mikulášová,
N. Tomášková,
J. Maderová and M. Kollárová
Purified malate dehydrogenase (MDH) of Streptomyces aureofaciens
was crystallized either in the absence or in the presence
of NADH or NADPH coenzymes by hanging-drop vapour-diffusion
method. An X-ray study has shown, that MDH crystals belong
to space group C2221 with unit-cell parameters
a = 53.2 Å, b = 104.6 Å, c = 520.0 Å, α
= β
= γ
= 90°, MDH-NADH crystals to space group C2 with unit-cell
parameters a = 51.5 Å, b = 51.5 Å, c = 256 Å,
α =
β =
γ =
90°, and MDH-NADPH crystals to space group C2221
with unit-cell parameters a = 72, Å b = 72 Å,
c = 520 Å, α
= β
= γ
= 90°. The crystal of native MDH diffracted to 2.1 Å
resolution.
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