Characterization of a Ribonucleic Acid Transcript from the Brook Trout (Salvelinus Fontinalis) Ovary with Structural Similarities to Mammalian Adipsin/Complement Factor D and Tissue Kallikrein, and the Effects of Kallikrein-Like Serine Proteases on Follicle Contraction1

Abstract

A 2.4-kilobase (kb) clone (kallikrein trout #14; KT-14) was isolated from a brook trout ovulatory cDNA library. KT-14 contains an open reading frame (ORF) of 768 base pairs (bp), presumably encoding a protein of 255 amino acids. The KT-14 cDNA also contains a 711 -bp 5' untranslated region and a 793-bp region downstream of the ORF that includes a 66-bp sequence repeated 12 times. The amino acid sequence of the KT-14 ORF is 41 % identical to that of porcine complement factor D and 33% identical to that of porcine pancreatic kallikrein. On Northern blots of ovarian tissue, KT-14 hybridized with four transcripts of 1.8, 2.4, 2.9, and 3.2 kb. While the 3.2- and 2.4-kb transcripts were present in the ovary prior to meiotic maturation, they were significantly up-regulated at ovulation and at 12 h postovulation, respectively. Antibodies constructed against the recombinant KT-14 protein recognized one 30-kDa immunogenic protein in ovarian tissue and fluid. This immunogenic protein was significantly elevated in the tissue by ovulation. Using a follicle weight loss bioassay, we provide indirect evidence that mammalian kallikrein and related serine proteases can stimulate brook trout follicle contraction. Thus, one possible function of the KT-14 protein may be the regulation of oocyte expulsion at ovulation.

The complement system in teleosts

“…Factor D, a relatively low molecular weight protein that is essential to the generation of the alternative pathway C3 convertase, has been purified from carp [30] and rainbow trout [27]. N-terminal sequencing of 25 residues of rainbow trout factor D showed 100% homology with a kallikrein-like molecule isolated from brook trout, Salvelinus fontinalis [27,31]. This structural similarity, together with the finding that antibodies directed against the kallikrein-like molecule reacted strongly with rainbow trout factor D (Lambvis et al, unpublished observations), suggests that the two proteins are the same molecule that is highly conserved among salmonids.…”

Cysteine protease inhibitor is specifically expressed in pre‐ and early‐vitellogenic oocytes from the brook trout periovulatory ovary*

“…A fusion protein was produced and puri®ed using an amylose af®nity column as previously described (Hajnik et al, 1998). A fraction with the highest protein content and the least amount of contaminating protein was chosen for immunization.…”

“…Immune serum was collected after exsanguination on day 52. IgGs were puri®ed from the immune serum and antibodies to MBP and copuri®ed E. coli proteins were removed as previously described (Hajnik et al, 1998).…”

“…Total protein was extracted from ovarian tissue and coelomic¯uid as previously described (Hajnik et al, 1998), and protein concentration was determined using the BCA protein assay (Pierce, Rockford, IL). For Western analysis, 30 mg of total protein from ovarian tissue or 20 mg coelomic¯uid protein were run on 10% reducing SDS±PAGE gels.…”

“…A strong antiserine protease activity was subsequently identi®ed in the trout coelomic¯uid that bathes ovulated eggs, and a signi®cant portion of this activity could be attributed to the presence of TOPs (Coffman and Goetz, 1998). In addition, cDNAs with sequences similar to a serine protease and an aspartic protease have been isolated from the brook trout ovary (Hajnik et al, 1998;Bobe and Goetz, 2001b). Cathepsin D (aspartic protease) has been cloned from the rainbow trout and the mRNA increases in the ovary at the beginning of vitellogenesis (Brooks et al, 1997).…”