Kohji Nomura

Purification and the primary structure of sperm-activating peptides from the jelly coat of sea urchin eggs

Abstract Two different peptides, which stimulate the respiration of spermatozoa, were purified from the jelly coat of sea urchin, Hemicentrotus pulcherrimus eggs. Analysis of the sequence by Edman degradation indicated that those sequences are Gly-Phe-Asp-Leu-Thr-Gly-Gly-Gly-Val and Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly.

Specificity and mode of action of the muscle-type protein-arginine deiminase

The primary and secondary specificities and mode of action of the muscle-type protein-arginine deiminase (PAD) were investigated using various derivatives of Arg and its homologues, as well as Arg-containing peptides by quantitative analyses of the reaction products on reverse-phase HPLC. The enzyme converted benzoyl-D-Arg-p-nitroanilide into its citrulline derivative at 18% of the rate of the L-isomer, while the D-Arg residues in peptides were not deiminated to a significant extent. This suggests that PAD does not have strict stereospecificity and it is dependent on the structure of the residues or groups on both sides of the target Arg residue. In contrast, the benzoyl-/-ethyl ester derivatives of homoarginine, alpha-amino-beta-guanidino-propionic acid, canavanine, and NG-methyl-Arg, exhibited poor PAD susceptibility, suggesting that the length and nature of the arm as exactly three CH2 groups, and the integrity of the guanidyl group are quite strict specificity determinants. The enzyme action on Arg residues in peptides depends greatly on their position in the sequence, and on the nature of the neighboring residues. For example, deimination of Arg residues situated at positions 1-3 from the NH2-terminus, except for those preceded by a carbobenzoxy- or benzoyl-group, were in most cases very slow, whereas those at the COOH-terminus were deiminated relatively faster. A single Arg residue sandwiched between two Pro residues was not deiminated at all, while a pair of Arg residues between two Pro were deiminated moderately. Consequently, PAD exhibited a variety of modes of action on more than one Arg residues in the peptides tested. The results suggest the applicability of PAD, albeit quite limited, for selective modification of certain Arg residues in peptides and proteins by appropriately controlling reaction time and several other parameters. The PADs mode of action was compared with those of three Arg-bond cleaving proteases.

Evidence for the involvement of calpain in cataractogenesis in Shumiya cataract rat (SCR)

The Shumiya cataract rat (SCR) is a hereditary cataract model in which lens opacity appears spontaneously in the nuclear and perinuclear portions at 11-12 weeks of age. It was found that the proteolysis of some crystallins and cytoskeletal proteins is significantly enhanced in cataractous SCR lenses. The calcium concentrations in cataractous lenses rise markedly with age as compared with control lenses and the autolytic product of calpain is also detected in cataractous lenses. In order to provide direct evidence for the involvement of calpain in the proteolytic modification of lens proteins, we developed antibodies exclusively specific to the proteolytic products of some lens proteins produced by the action of calpain and analyzed their degradation during cataractogenesis in SCR by Western blotting and immunohistochemical staining. The results demonstrate that calpain participates in the proteolytic modification of lens proteins, at least alpha-crystallin (A and B chain), betaB1-crystallin, and alpha-fodrin. The proteolytic products formed by the action of calpain on these proteins are detected in cataractous lenses of SCR as young as 8 weeks of age and accumulate with age. It was also found that betaB1-crystallin, originally a soluble protein, is converted to an insoluble form by limited calpain proteolysis. The chaperon-like activity of alpha-crystallin from control lens is markedly reduced by calpain proteolysis in vitro, and alpha-crystallin in opaque lens that has already undergone proteolysis by calpain shows significantly reduced chaperon-like activity. Immunohistochemical studies reveal that the area where the calpain-mediated alpha-crystallin proteolysis is in progress coincides well with the area developing and destined to develop the opacification. These results strongly suggest that calpain may contribute to lens opacification during cataract formation in SCR.

Synthetic study on the structure-activity relationship of Sperm Activating Peptides from the jelly coat of sea urchin eggs

Various analogue peptides with substitution and deletion of amino acid residues have been synthesized by liquid phase method for Sperm Activating Peptides from the jelly coat of sea urchin eggs. The deletion of C-terminal Gly reduced the activity to about 1/3000, while removal of N-terminal Gly reduced the activity to 1/10. The residues Ser5 and Asp3 were replaced by Lys without significant loss of activity. Substitution of Phe2 by Gly, Ala or Pro markedly reduced the activity by the factor of 10(4)-10(6), in contrast to Tyr-substitution retaining almost full activity, indicating the essential role of the aromatic residue in exerting the activity. Substitutions, Asp3 to Glu and Gly10 to Pro, increased the activity 5-fold and 500-fold, respectively.

Some more speract derivatives associated with eggs of sea urchins Pseudocentrotus depressus, Strongylocentrotus purpuratus, Hemicentrotus pulcherrimus and Anthocidaris crassispina

1. Fourteen peptides were isolated from the egg jelly of sea urchins, Pseudocentrotus depressus, Strongylocentrotus purpuratus, Hemicentrotus pulcherrimus and Anthocidaris crassispina and their amino acid sequences were determined. 2. The peptides stimulated H. pulcherrimus sperm respiration one half-maximally at about 8-60 pM. 3. Addition of speract to intact spermatozoa of P. depressus, H. pulcherrimus and A. crassispina resulted in the appearance of a newly stained protein (Mr 128,000 for P. depressus, Mr 128,000 for H. pulcherrimus and Mr 131,000 for A. crassispina) on sodium dodecyl sulfate-polyacrylamide gels.

Structure and action of sperm activating peptides from the egg jelly of a sea urchin, Anthocidaris crassispina.

Three Sperm Activating Peptides (SAPs) have been isolated to homogeneity from the jelly coat of the eggs of a Japanese sea urchin, Anthocidaris crassispina, and two of them have been sequenced. At pH 6.8 they can stimulate the sperm respiration 20-30 fold, to the level in normal seawater (pH 8.2), and the half-maximal activation was achieved by SAPs as low as around 100 pM. The stimulative activity was both pH- and Na+-dependent. The chymotryptic fragments (res. 3-10) were 10(4)-10(5) times less active, and the thermolytic fragment (res. 4-10) was 10(6) times less active than the parent SAP. CD spectra of SAPs indicate that they have unordered structure in aqueous solution.

Stereo-specific inhibition of sea urchin envelysin (hatching enzyme) by a synthetic autoinhibitor peptide with a cysteine-switch consensus sequence

Inhibition of envelysin, a metalloproteinase which dissolves the fertilization envelope of sea urchin embryo, was studied using a synthetic autoinhibitor peptide, Ac‐Pro‐Arg‐Cys‐Gly‐Val‐Pro‐Asp‐Val‐NH2, with a ‘cysteine‐switch’ consensus sequence. Although its effect is reversible, the hatching of sea urchin embryos was effectively delayed by 0.5 mM of the peptide. When α1‐proteinase inhibitor was used as the substrate, envelysin was inhibited by the autoinhibitor and an Ala6 analogue, but not by a d‐Cys3 analogue. However, envelysin was weakly inhibited by both d‐ and l‐cysteines to the same extent. Snake venom α‐protease exhibited cleavage and inhibition behavior similar to envelysin with a little weaker stereo‐specificity. The results suggest that the coordination of the autoinhibitor Cys residue with the envelysin active site Zn is established only after the amino acid residues on both sides of the Cys residue get into an appropriate interaction with the catalytic site residues, and that the precise orientation of the cysteine SH group is essential. By contrast, thermolysin was weakly inhibited by the three peptide non‐stereo‐specifically. Furthermore, thermolysin cleaved the autoinhibitor at the Cys3 Gly4 bond when incubated without substrate.

A novel group of sperm activating peptides from the sea urchin Glyptocidaris crenularis.

1. Six new sperm activating peptides were purified from the egg jelly of the sea urchin Glyptocidaris crenularis and their amino acid sequences were determined as follows: Ser-Ala-Lys-Leu-Cys-Pro-Gly-Gly-Asn-Cys-Val, Lys-Leu-Cys-Pro-Gly-Gly-Asn-Cys-Val, Leu-Cys-Pro-Gly-Gly-Asn-Cys-Val, Ser-Phe-Lys-Leu-Cys-Pro-Gly-Gly-Gln-Cys-Val, Lys-Leu-Cys-Pro-Gly-Gly-Gln-Cys-Val and Leu-Cys-Pro-Gly-Gly-Gln-Cys-Val. 2. The peptides were specific for G. crenularis spermatozoa and caused significant increases of sperm respiration rates and sperm cyclic nucleotide concentrations at concentrations as low as 10(-10) M. 3. The addition of the peptides to intact spermatozoa resulted in the change of the apparent mol. wt of a sperm protein from 195,000 to 200,000.

A species-specific sperm-activating peptide from the egg jelly of the sea urchin Diadema setosum

1. A species-specific sperm-activating peptide was isolated from the egg jelly of the sea urchin Diadema setosum and the amino acid sequence was determined as follows: (formula; see text). 2. The peptide caused significant increases of respiration rates and cyclic nucleotide concentrations in D. setosum spermatozoa as low as 10(-9) M. 3. The addition of the peptide to D. setosum spermatozoa resulted in the appearance of a newly stained protein (mol. wt 128,000) on sodium dodecyl sulfate-polyacrylamide gels.

A mRNA for Membrane Form of Guanylyl Cyclase Is Expressed Exclusively in the Testis of the Sea Urchin Hemicentrotus pulcherrimus

Abstract A cDNA clone encoding the membrane form of guanylyl cyclase was isolated from a Hemicentrotus pulcherrimus testis cDNA library and its nucleotide sequence was determined. The cDNA was 4123 bp long and an open reading frame predicted a protein of 1125 amino acids including an apparent signal peptide of 21 residues; a single transmembrane domain of 25 amino acids which divides the mature protein into an amino-terminal, extracellular domain of 485 amino acids and a carboxyl-terminal, intracellular domain of 594 amino acids. Three potential N-linked glycosylation sites were present in the extracellular domain. Northern blot analysis of poly(A)+RNA from testes, ovaries, eggs and embryos at various developmental stages showed that the cDNA encoding guanylyl cyclase hybridized to a mRNA of 4.4 kb from the testes. We developed a large scale purification method of the phosphorylated (131 kDa) and dephosphory-lated (128 kDa) forms of the membrane-bound guanylyl cyclase from H. pulcherrimus spermatozoa. The purified 131 kDa and 128 kDa forms of the guanylyl cyclase contained 26.0 ± 1.3 and 4.3 ± 0.7 moles of phosphate per mol protein (mean ± S.D.; n=6), respectively. The amino-terminal amino acids of both the 131 kDa and 128 kDa forms of the guanylyl cyclase could not be detected, suggesting that they were blocked.