Speract

The isolation, characterization, and expression of a full-length cDNA encoding the human T cell glycoprotein CD6 is described. COS cells transfected with the CD6 clone express a 90-kD protein that reacts with all available anti-CD6 monoclonal antibodies. RNA blot hybridization analysis indicates that CD6 transcripts are predominantly restricted to cells in the T lineage. The predicted CD6 sequence is 468 amino acids long, with the typical features of a type I integral membrane protein. The cytoplasmic domain of CD6 contains two serine residues, one or both of which are substrates for phosphorylation during T cell activation. The extracellular domain of CD6 is significantly related to the extracellular domain of the human and mouse T cell antigen CD5, the cysteine-rich domain of the bovine and mouse type I macrophage scavenger receptor, the extracellular domain of the sea urchin spermatozoa protein that crosslinks the egg peptide speract, the mammalian complement factor 1, and the human lung tumor antigen L3. These molecules, therefore, constitute a new gene superfamily that is well conserved across species boundaries.

At an extracellular pH of 6.6, a peptide (resact) isolated from the egg jelly of Arbacia punctulata increased the respiration rates of A. punctulata spermatozoa but did not activate sperm cells from Lytechinus pictus. In contrast, speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly), elevated the respiration rates of L. pictus but not A. punctulata spermatozoa. At normal seawater pH (7.6-8.0) egg jelly from A. punctulata, or egg jelly from L. pictus purified free of speract, inhibited L. pictus sperm respiration rates. Similarly, the egg jelly from L. pictus inhibited the respiration rates of A. punctulata spermatozoa. The jelly component responsible for the inhibition of respiration was nondialyzable. The inhibition of respiration induced by jelly could be reversed by the addition of speract to L. pictus spermatozoa and by the addition of resact to A. punctulata spermatozoa. Speract stimulated L. pictus sperm respiration half-maximally at about 1 nM in the presence of either heterologous or homologous (speract-free) jelly. Monensin A, an ionophore which elevates sperm intracellular pH, reversed the jelly inhibition of respiration. These results demonstrate that two peptides associated with eggs (speract and resact) can stimulate sperm motility and metabolism in the face of inhibitory components present in the egg jelly.

GGGYG-resact (Gly-Gly-Gly-Tyr-Gly-Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg -Leu-NH2) was synthesized and shown to possess the same respiration-stimulating activity and receptor-binding ability as resact. The incubation of intact sperm cells with radioiodinated peptide, 125I-GGGYG-resact, and the chemical cross-linking reagent, disuccinimidyl suberate, resulted in the appearance of a single, major radioactive band of apparent molecular weight 160,000 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The interaction was specific since 150 nM nonradioactive resact but not speract (200 nM) blocked formation of the radioactive band. The radioactive, cross-linked protein co-migrated with 32P-labeled guanylate cyclase and could be immunoprecipitated with a polyclonal antibody raised in rabbits against the sperm guanylate cyclase. The incubation of intact cells with NH4Cl resulted in the partial dephosphorylation of guanylate cyclase and a change in its apparent molecular weight from 160,000 to 150,000; NH4Cl also caused the same conversion in the apparent molecular weight of the cross-linked protein. These data demonstrate that an analogue of resact can be covalently coupled to guanylate cyclase with the specificity predicted for the peptide receptor.