Rat testin is a newly identified component of the junctional complexes in various tissues whose mRNA is predominantly...

Testin I and testin II are the two molecular variants of testin that are synthesized and secreted by Sertoli cells in vitro. N-Terminal and partial internal amino acid sequence analysis of testin I and testin II reveals that these molecules are identical with the exception that testin II has three extra N-terminal amino acids of TAP compared to testin I. Studies using immunohistochemistry suggested that testin is a component of the specialized junctional complexes in the seminiferous epithelium and other tissues. Immunoreactive testin is localized not only at Sertoli-Sertoli and Sertoli-germ cell junctions, but also at sites of similar junctions in the liver, epididymis, kidney, and intestine. Other physiological studies have shown that the secretion of testin is tightly coupled to the presence of germ cells. In view of its possible role in germ cell development and its unique localization in the cell junction, the purpose of the present study was to determine the structure of testin by sequencing its full-length cDNA. Two synthetic degenerate oligonucleotides based on the N-terminal and an internal amino acid sequence were used for polymerase chain reaction (PCR) to obtain a 289-bp cDNA fragment. This PCR product was subsequently used to isolate a 1371-bp cDNA from a cDNA expression library constructed from Sertoli cell poly(A) RNA. This cDNA coded for a 333 amino acid peptide that starts with an ATG initiation codon from the 5' end and ends with a TGA termination codon located 245 nucleotides before the polyadenylation site. The deduced amino acid sequence indicates that testin contains a 16 amino acid signal peptide with two possible cleavage sites that yield 314 and 317 amino acids for testin I and testin II with calculated molecular weights of 36,029 and 36,299, respectively. Comparison of the entire coding region of testin with existing sequences at Genbank, EMBL, and Protein Identification Resource indicates that testin shares 58%, 57.4%, and 61% identity with rat, mouse, and human cathepsin L at the amino acid level, respectively. The positions of all of the 7 Cys residues and 8 of the 10 Trp residues in testin are conserved with respect to those present in cathepsin L. It is noted that Cys-122 in the predicted active site of cathepsin L was replaced with Ser-122 in testin. In view of the striking primary sequence homology between testin and cathepsin L, we assayed the proteolytic activity of testin using conditions known to activate cathepsin L.(ABSTRACT TRUNCATED AT 400 WORDS)