Three genes expressing Kunitz domains in the epididymis are related to genes of WFDC-type protease inhibitors and semen coagulum proteins in spite of lacking similarity between their protein products

Abstract

Background: We have previously identified a locus on human chromosome 20q13.1, encompassing related genes of postulated WFDC-type protease inhibitors and semen coagulum proteins. Three of the genes with WFDC motif also coded for the Kunitz-type protease inhibitor motif. In this report, we have reinvestigated the locus for homologous genes encoding Kunitz motif only. The identified genes have been analyzed with respect to structure, expression and function.

Results: We identified three novel genes; SPINT3, SPINT4 and SPINT5, and the structure of their transcripts were determined by sequencing of DNA generated by rapid amplification of cDNA ends. Each gene encodes a Kunitz domain preceded by a typical signal peptide sequence, which indicates that the proteins of 7.6, 8.7, and 9.7 kDa are secreted. Analysis of transcripts in 26 tissues showed that the genes predominantly are expressed in the epididymis. The recombinantly produced proteins could not inhibit the amidolytic activity of trypsin, chymotrypsin, plasmin, thrombin, coagulation factor Xa, elastase, urokinase and prostate specific antigen, whereas similarly made bovine pancreatic trypsin inhibitor (BPTI) had the same bioactivity as the protein isolated from bovine pancreas.

Conclusions: The similar organization, chromosomal location and site of expression, suggests that the novel genes are homologous with the genes of WFDC-type protease inhibitors and semen coagulum proteins, despite the lack of similarity in primary structure of their protein products. Their restricted expression to the epididymis suggests that they could be important for male reproduction. The recombinantly produced proteins are presumably bioactive, as demonstrated with similarly made BPTI, but may have a narrower spectrum of inhibition, as indicated by the lacking activity against eight proteases with differing specificity. Another possibility is that they have lost the protease inhibiting properties, which is typical of Kunitz domains, in favor of hitherto unknown functions.

Figure 1

Potential Kunitz inhibitors at the human WFDC locus. The two DNA-strands of the telomeric sublocus of the WFDC/Semenogelin locus on human chromosome 20 are displayed as horizontal bars. The location of genes is indicated by thicker bars with arrow heads above or below, which illustrate WFDC domains in white, Kunitz domains in black and signal peptide in grey.

Figure 2

Structure of SPINT3 and SPINT4. The nucleotide sequences of transcripts are given with translation written above for (A) SPINT3 and (B) SPINT4. The arrows indicate location of the predicted signal peptide cleavage sites. The two Cys in SPINT4, which might form an unconventional disulphide bond, are boxed. The nucleotides present in the longer SPINT4 transcript, but not in the shorter, are italicized.

Figure 3

Conservation of SPINT5. The mouse and human SPINT5 transcripts were aligned with the computer program ALIGN. The nucleotide sequences that correspond to exon 1-3 of mouse and human SPINT5 are shown with identities indicated by vertical bars and gapped nucleotides by dashes. The second exon is shaded, with non-aligning nucleotides in the human gene inserted below; note the CAG at the end of the insertion that might indicate splice acceptor site, and the arrow that indicates starting nucleotide of the shorter 5' RACE product. The 3' ends of mouse Spint5, human SPINT5, and alternative 3' transcript of human SPINT5 are given at the bottom of the figure with poly-adenylation signals written in bold; location of introns in the alternative SPINT5 3' end are indicated with opposing arrows.

Figure 4

Protease inhibition with recombinant BPTI. (A) Serial dilutions of recombinant BPTI were incubated with 20 nM bovine chymotrypsin (■), 50 nM human plasmin (▼), and 5 nM bovine trypsin (▲). (B) Recombinant BPTI (filled symbols) and BPTI isolated from bovine pancreas (open symbols) were incubated with 10 nM (circles) and 20 nM (squares) bovine chymotrypsin. Residual amidolytic activity was measured and compared with samples without added inhibitor

Figure 5

SDS-PAGE. Purified recombinant proteins were separated by SDS-PAGE and then silver stained. Masses of the molecular standard proteins in kDa are given to the left.

Figure 6

Conservation of the Kunitz domain. Exons encoding Kunitz domains were translated and the resulting peptide sequences were aligned using CLUSTALW. The amino acid residues homologous with those of the peptidase binding loop (P3-P'3) in BPTI are framed and the P1 position is shaded. Positions with mutated Cys in SPINT4 and WFDC6 are framed and shaded.