Trypsin/Acrosin Inhibitor Activity of Rat and Guinea Pig Caltrin Proteins. Structural and Functional Studies1

Abstract

Dramatic inhibition of trypsin activity by rat caltrin and guinea pig caltrin I was spectrophotometrically demonstrated using the artificial substrate benzoylarginyl ethyl ester. Approximately 6% and 21% of residual proteolytic activity was recorded after preincubating the enzyme with 0.22 and 0.27 microM rat caltrin and guinea pig caltrin I, respectively. Reduction and carboxymethylation of the cysteine residues abolished the inhibitor activity of both caltrin proteins. Rat caltrin and guinea pig caltrin I show structural homology with secretory trypsin/acrosin inhibitor proteins isolated from boar and human seminal plasma and mouse seminal vesicle secretion and share a fragment of 13 amino acids of almost identical sequence (DPVCGTDGH/K/ITYG/AN), which is also present in the structure of Kazal-type trypsin inhibitor proteins from different mammalian tissues. Bovine, mouse, and guinea pig caltrin II, three caltrin proteins that have no structural homology with rat caltrin or guinea pig caltrin I, lack trypsin inhibitor activity. Rat caltrin, guinea pig caltrin I, and the mouse seminal vesicle trypsin inhibitor protein P12, which also inhibits Ca(2+) uptake into epididymal spermatozoa (mouse caltrin I), bound specifically to the sperm head, on the acrosomal region, as detected by indirect immunofluorescence. They also inhibited the acrosin activity in the gelatin film assay. Caltrin I may play an important role in the control of sperm functions such as Ca(2+) influx in the acrosome reaction and activation of acrosin and other serine-proteases at the proper site and proper time to ensure successful fertilization.

SPINK3 modulates mouse sperm physiology through the reduction of nitric oxide level independently of its trypsin inhibitory activity

“…It can inhibit trypsin in vitro (Fig. 2) as shown with purified P12 (Lai et al 1994, Winnica et al 2000. It also inhibits acrosin (Winnica et al 2000), but the target protease in sperm is still unknown.…”

“…2) as shown with purified P12 (Lai et al 1994, Winnica et al 2000. It also inhibits acrosin (Winnica et al 2000), but the target protease in sperm is still unknown. Recently, a membrane-bound serine protease, TESPL, has been proposed to be the SPINK3 anchoring protein because interaction between these proteins has been demonstrated by yeast two-hybrid assay (Ou et al 2010).…”

“…caltrin I binds to the head of epididymal sperm and prevents AR during in vitro capacitation (Winnica et al 2000); while caltrin II binds to the principal portion of the tail and delays sperm hyperactivation . Caltrin II has no structural homology with caltrin I (Winnica et al 2000) even though its gene conserves a whey acidic protein (WAP) domain, which is present in serine protease inhibitors (Furutani et al 2004).…”

“…Caltrin II has no structural homology with caltrin I (Winnica et al 2000) even though its gene conserves a whey acidic protein (WAP) domain, which is present in serine protease inhibitors (Furutani et al 2004). …”

Happily at Work

“…Reducing the cystine disulfide bonds of rat caltrin and carboxymethylating the protein diminishes, but does not eliminate, the effect on calcium transport. The locations of the disulfide bonds are known (52). In the case of mouse caltrin, which contains 7 cysteine residues, the protein appears to be a disulfide dimer formed between the odd cysteines.…”