Distinction of sperm-binding site and reactive site for trypsin inhibition on p12 secreted from the accessory sex glands of male mice.
Six variants of P12, a Kazal-type trypsin inhibitor in the secretion of male mouse accessory sexual glands, were made using single-site mutations including R19L, Y21V, D22G, R43G, K44S, and R45T, based on one-letter-code mutation of amino acids. The other two variants, Nd10 and Cd8, were made using the deletion of 10 and 8 residues from the N- and C-terminals, respectively. Their CD profiles revealed maintenance of the P12 conformation in the seven variants, excluding Cd8, which became unfolded. Only R19L entirely lost the ability while the other variants were as strong as P12 in inhibiting the trypsin digestion of N-benzoyl-Phe-Val-Arg 7-amido-4-methylcoumarin. The immunocytochemical results demonstrated that D22G and Cd8 failed to bind to sperm, Y21V very weakly did so, and the other variants retained their sperm-binding abilities. Concomitantly, the immunocytochemical stainability of each ligand was parallel to its inhibitory effect on 125I-P12-sperm binding, and a synthetic oligopeptide corresponding to residues 18-24 of P12 was able to inhibit P12-sperm binding. The data together concluded that R19 was essential for protease inhibition and D22 and/or Y21 mainly being responsible for the binding of P12 to sperm. The steric arrangement of R19, Y21, and D22 on the tertiary structure of P12 is discussed.
Luo
Ching-Wei
CW
Institute of Biochemical Sciences, College of Science, National Taiwan University, Taipei 106, Taiwan.
Lin
Han-Jia
HJ
Gopinath
S C B
SC
Chen
Yee-Hsiung
YH
D000595:Amino Acid Sequence; D000818:Animals; D021461:Animals, Outbred Strains; D001665:Binding Sites; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D017353:Gene Deletion; D014644:Genetic Variation; D005837:Genitalia, Male / Q000378:metabolism*; D006023:Glycoproteins; D008297:Male; D051379:Mice; D009154:Mutation; D029584:Prostatic Secretory Proteins; D011487:Protein Conformation; D013094:Spermatozoa / Q000378:metabolism*; D014359:Trypsin Inhibitor, Kazal Pancreatic; D014361:Trypsin Inhibitors / Q000494:pharmacology
D004268:DNA-Binding Proteins; D006023:Glycoproteins; D029584:Prostatic Secretory Proteins; C487134:Spink1 protein, mouse; D014361:Trypsin Inhibitors; D014359:Trypsin Inhibitor, Kazal Pancreatic