Abstract
Interrogation of the public expressed sequence tag (EST) data base with the sequence of preproaprotinin identified ESTs encoding two potential new members of the Kunitz family of serine protease inhibitors. Through reiterative interrogation, an EST contig was obtained, the consensus sequence from which encoded both of the novel Kunitz domains in a single open reading frame. This consensus sequence was used to direct the isolation of a full-length cDNA clone from a placental library. The resulting cDNA sequence predicted a 252-residue protein containing a putative NH2-terminal signal peptide followed sequentially by each of the two Kunitz domains within a 170-residue ectodomain, a putative transmembrane domain, and a 31-residue hydrophilic COOH terminus. The gene for this putative novel protein was mapped by use of a radiation hybrid panel to chromosome 19q13, and Northern analysis showed that the corresponding mRNA was expressed at high levels in human placenta and pancreas and at lower levels in brain, lung, and kidney. An endogenous soluble form of this protein, which was designated as placental bikunin, was highly purified from human placenta by sequential kallikrein-Sepharose affinity, gel filtration, and C18 reverse-phase chromatography. The natural protein exhibited the same NH2 terminus as predicted from the cloned cDNA and inhibited trypsin, plasma kallikrein, and plasmin with IC50 values in the nanomolar range.
Highlights
The Kunitz [1, 2], Kazal [2], Serpin [3], and mucus [4] families of biological serine protease inhibitors play a vital role in the spatial and temporal regulation of in vivo proteolysis
R35464 and R74593 established that the 3Ј nucleotide sequence flanking the Kunitz domain encoded in R35464 was identical to the 5Ј sequence flanking the Kunitz domain encoded in R74593
Several overlapping expressed sequence tag (EST) were obtained upon reinterrogation with R35464 and R74593, which were in turn used to reinterrogate data base of ESTs (dbEST)
Summary
The Kunitz [1, 2], Kazal [2], Serpin [3], and mucus [4] families of biological serine protease inhibitors play a vital role in the spatial and temporal regulation of in vivo proteolysis. The physiologic function of aprotinin is uncertain, it is a potent inhibitor of several serine proteases, and its potency against kallikrein and plasmin [5] may be relevant to its clinical mode of action [5, 6], in the reduction of perioperative blood loss. A human functional homolog of aprotinin has not been identified, several larger human proteins containing one or more Kunitz domains are known. To identify novel human homologs of aprotinin we employed a bioinformatic approach that exploited the rapidly expanding human expressed sequence tags (ESTs) data base [17, 18]. This resulted in the discovery of a novel human gene product designated as placental bikunin
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