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1.
Positive selection is a general phenomenon in the evolution of abalone sperm lysin 总被引:36,自引:21,他引:15
Lysin is a 16kDa acrosomal protein used by abalone sperm to create a hole
in the egg vitelline envelope (VE). The interaction of lysin with the VE is
species-selective and is one step in the multistep fertilization process
that restricts heterospecific (cross-species) fertilization. For this
reason, the evolution of lysin could play a role in establishing prezygotic
reproductive isolation between species. Previously, we sequenced sperm
lysin cDNAs from seven California abalone species and showed that positive
Darwinian selection promotes their divergence. In this paper an additional
13 lysin sequences are presented representing species from Japan, Taiwan,
Australia, New Zealand, South Africa, and Europe. The total of 20 sequences
represents the most extensive analysis of a fertilization protein to date.
The phylogenetic analysis divides the sequences into two major clades, one
composed of species from the northern Pacific (California and Japan) and
the other composed of species from other parts of the world. Analysis of
nucleotide substitution demonstrates that positive selection is a general
process in the evolution of this fertilization protein. Analysis of
nucleotide and codon usage bias shows that neither parameter can account
for the robust data supporting positive selection. The selection pressure
responsible for the positive selection on lysin remains unknown.
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2.
Mitochondrial DNA and bindin gene sequence evolution among allopatric species of the sea urchin genus Arbacia 总被引:3,自引:1,他引:2
Sea urchins of the genus Arbacia (order Stirodonta) have discontinuous
allopatric distributions ranging over thousands of kilometers.
Mitochondrial DNA (mtDNA) sequences were used to reconstruct phylogenetic
relationships of four Arbacia species and their geographic populations.
There is little evidence of genetic structuring of populations within
species, except in two cases at range extremes. The mtDNA sequence
differentiation between species suggests that divergence occurred about 4-9
MYA. Gene sequences encoding the sperm protein bindin and its intron were
obtained and compared with the mtDNA phylogeny. Sea urchins among the
well-studied echinoid order Camarodonta, with degrees of mtDNA divergence
similar to those of Arbacia species, are known to have remarkable variation
in bindin. However, in Arbacia, little variation in deduced amino acid
sequences of bindin was found, indicating that purifying selection acts on
the protein. In contrast, bindin intron sequences showed much
differentiation, including numerous insertion/deletions. Fertilization
experiments performed between a divergent pair of Arbacia species from the
Atlantic and Pacific Oceans revealed no evidence of blocks to gamete
recognition. In Arbacia, fertilization specificities may have evolved
relatively slowly as a result of extensive gene flow within species,
greater functional constraint on the bindin polypeptide, or reduced
selective pressure for species recognition in singly occurring species.
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3.
Females of the sea urchin Strongylocentrotus purpuratus differ in the structures of their egg jelly sulfated fucans 总被引:1,自引:0,他引:1
The egg jelly coats of sea urchins contain sulfated fucans which bind to a
sperm surface receptor glycoprotein to initiate the signal transduction
events resulting in the sperm acrosome reaction. The acrosome reaction is
an ion channel regulated exocytosis which is an obligatory event for sperm
binding to, and fusion with, the egg. Approximately 90% of individual
females of the sea urchin Strongylocentrotus purpuratus spawned eggs having
only one of two possible sulfated fucan electrophoretic isotypes, a slow
migrating (sulfated fucan I), or a fast migrating (sulfated fucan II)
isotype. The remaining 10% of females spawned eggs having both sulfated
fucan isotypes. The two sulfated fucan isotypes were purified from egg
jelly coats and their structures determined by NMR spectroscopy and
methylation analysis. Both sulfated fucans are linear polysaccharides
composed of 1-->3-linked alpha-L-fucopyranosyl units. Sulfated fucan I
is entirely sulfated at the O -2 position but with a heterogeneous
sulfation pattern at O -4 position. Sulfated fucan II is composed of a
regular repeating sequence of 3 residues, as follows: [3-alpha-L-Fuc p -
2,4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)-
1]n. Both purified sulfated fucans have approximately equal potency in
inducing the sperm acrosome reaction. The significance of two structurally
different sulfated fucans in the egg jelly coat of this species could
relate to the finding that the sperm receptor protein which binds sulfated
fucan contains two carbohydrate recognition modules of the C-type lectin
variety which differ by 50% in their primary structure.
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4.
Expression, purification, and characterization of recombinant cyanovirin-N for vaginal anti-HIV microbicide development 总被引:7,自引:0,他引:7
Colleluori DM Tien D Kang F Pagliei T Kuss R McCormick T Watson K McFadden K Chaiken I Buckheit RW Romano JW 《Protein expression and purification》2005,39(2):229-236
Cyanovirin-N (CV-N) is a prokaryotic protein under development as a topical anti-HIV microbicide, an urgent and necessary approach to prevent HIV transmission in at-risk populations worldwide. We have expressed recombinant CV-N as inclusion bodies in the cytoplasm of Escherichia coli. A purification scheme has been developed that exploits the physicochemical properties of this protein, in particular its stability in a harsh inclusion body purification scheme. Under the conditions developed, this system yields 140 mg of highly purified CV-N per liter of high-density cell culture, which represents a 14-fold increase over the best recombinant CV-N yield reported to date. This purification scheme results in monomeric CV-N as analyzed by SDS-PAGE, isoelectric focusing, and reverse phase- and size exclusion-HPLC. This recombinantly expressed and refolded CV-N binds to gp120 with nanomolar affinity and retains its potent anti-HIV activities in cell-based assays. The expression and purification system described herein provides a better means for the mass production of CV-N for further microbicide development. 相似文献
5.
Mechanistic and metabolic inferences from the binding of substrate analogues and products to arginase 总被引:2,自引:0,他引:2
Cox JD Cama E Colleluori DM Pethe S Boucher JL Mansuy D Ash DE Christianson DW 《Biochemistry》2001,40(9):2689-2701
Arginase is a binuclear Mn(2+) metalloenzyme that catalyzes the hydrolysis of L-arginine to L-ornithine and urea. X-ray crystal structures of arginase complexed to substrate analogues N(omega)-hydroxy-L-arginine and N(omega)-hydroxy-nor-L-arginine, as well as the products L-ornithine and urea, complete a set of structural "snapshots" along the reaction coordinate of arginase catalysis when interpreted along with the X-ray crystal structure of the arginase-transition-state analogue complex described in Kim et al. [Kim, N. N., Cox, J. D., Baggio, R. F., Emig, F. A., Mistry, S., Harper, S. L., Speicher, D. W., Morris, Jr., S. M., Ash, D. E., Traish, A. M., and Christianson, D. W. (2001) Biochemistry 40, 2678-2688]. Taken together, these structures render important insight on the structural determinants of tight binding inhibitors. Furthermore, we demonstrate for the first time the structural mechanistic link between arginase and NO synthase through their respective complexes with N(omega)-hydroxy-L-arginine. That N(omega)-hydroxy-L-arginine is a catalytic intermediate for NO synthase and an inhibitor of arginase reflects the reciprocal metabolic relationship between these two critical enzymes of L-arginine catabolism. 相似文献
6.
Arginases catalyze the hydrolysis of L-arginine to yield L-ornithine and urea. Recent studies indicate that arginases, both the type I and type II isozymes, participate in the regulation of nitric oxide production by modulating the availability of arginine for nitric oxide synthase. Due to the reciprocal regulation between arginase and nitric oxide synthase, arginase inhibitors have therapeutic potential in treating nitric oxide-dependent smooth muscle disorders, such as erectile dysfunction. We demonstrate the competitive inhibition of the mitochondrial human type II arginase by N(omega)-hydroxy-L-arginine, the intermediate in the reaction catalyzed by nitric oxide synthase, and its analogue N(omega)-hydroxy-nor-L-arginine, with K(i) values of 1.6 microM and 51 nM at pH 7.5, respectively. We also demonstrate the inhibition of human type II arginase by the boronic acid-based transition-state analogues 2(S)-amino-6-boronohexanoic acid (ABH) and S-(2-boronoethyl)-L-cysteine (BEC), which are known inhibitors of type I arginase. At pH 7.5, both ABH and BEC are classical, competitive inhibitors of human type II arginase with K(i) values of 0.25 and 0.31 microM, respectively. However, at pH 9.5, ABH and BEC are slow-binding inhibitors of the enzyme with K(i) values of 8.5 and 30 nM, respectively. The findings presented here indicate that the design of arginine analogues with uncharged, tetrahedral functional groups will lead to the development of more potent inhibitors of arginases at physiological pH. 相似文献
7.
8.
Cama E Colleluori DM Emig FA Shin H Kim SW Kim NN Traish AM Ash DE Christianson DW 《Biochemistry》2003,42(28):8445-8451
Arginase is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of l-arginine to form l-ornithine and urea. The X-ray crystal structure of a fully active, truncated form of human arginase II complexed with a boronic acid transition state analogue inhibitor has been determined at 2.7 A resolution. This structure is consistent with the hydrolysis of l-arginine through a metal-activated hydroxide mechanism. Given that human arginase II appears to play a role in regulating l-arginine bioavailability to NO synthase in human penile corpus cavernosum smooth muscle, the inhibition of human arginase II is a potential new strategy for the treatment of erectile dysfunction [Kim, N. N., Cox, J. D., Baggio, R. F., Emig, F. A., Mistry, S., Harper, S. L., Speicher, D. W., Morris, S. M., Ash, D. E., Traish, A. M., and Christianson, D. W. (2001) Biochemistry 40, 2678-2688]. Since NO synthase is found in human clitoral corpus cavernosum and vagina, we hypothesized that human arginase II is similarly present in these tissues and functions to regulate l-arginine bioavailability to NO synthase. Accordingly, hemodynamic studies conducted with a boronic acid arginase inhibitor in vivo are summarized, suggesting that the extrahepatic arginase plays a role in both male and female sexual arousal. Therefore, arginase II is a potential target for the treatment of male and female sexual arousal disorders. 相似文献
9.
Colleluori DM Reczkowski RS Emig FA Cama E Cox JD Scolnick LR Compher K Jude K Han S Viola RE Christianson DW Ash DE 《Archives of biochemistry and biophysics》2005,444(1):15-26
Rat liver arginase (arginase I) is potently inactivated by diethyl pyrocarbonate, with a second-order rate constant of 113M(-1)s(-1) for the inactivation process at pH 7.0, 25 degrees C. Partial protection from inactivation is provided by the product of the reaction, l-ornithine, while nearly complete protection is afforded by the inhibitor pair, l-ornithine and borate. The role of H141 has been probed by mutagenesis, chemical modulation, and X-ray diffraction. The hyper-reactivity of H141 towards diethyl pyrocarbonate can be explained by its proximity to E277. A proton shuttling role for H141 is supported by its conformational mobility observed among the known arginase structures. H141 is proposed to serve as an acid/base catalyst, deprotonating the metal-bridging water molecule to generate the metal-bridging hydroxide nucleophile, and by protonating the amino group of the product to facilitate its departure. 相似文献
10.
Human type II arginase, which is extrahepatic and mitochondrial in location, catalyzes the hydrolysis of arginine to form ornithine and urea. While type I arginases function in the net production of urea for excretion of excess nitrogen, type II arginases are believed to function primarily in the net production of ornithine, a precursor of polyamines, glutamate, and proline. Type II arginases may also regulate nitric oxide biosynthesis by modulating arginine availability for nitric oxide synthase. Recombinant human type II arginase was expressed in Escherichia coli and purified to apparent homogeneity. The Km of arginine for type II arginase is approximately 4.8 mM at physiological pH. Type II arginase exists primarily as a trimer, although higher order oligomers were observed. Borate is a noncompetitive inhibitor of the enzyme, with a Kis of 0.32 mM and a Kii of 0.3 mM. Ornithine, a product of the reaction catalyzed by arginase and a potent inhibitor of type I arginase, is a poor inhibitor of the type II isozyme. The findings presented here indicate that isozyme-selectivity exists between type I and type II arginases for binding of substrate and products, as well as inhibitors. Therefore, inhibitors with greater isozyme-selectivity for type II arginase may be identified and utilized for the therapeutic treatment of smooth muscle disorders, such as erectile dysfunction. 相似文献