Through the analysis of the porcine gene encoding the elastase inhibitor elafin, we demonstrated that there are at least three closely related members of the elafin family, and their genes have arisen by accelerated evolution. A porcine genomic DNA library was screened with a previously cloned human elafin cDNA probe, and several positive clones were obtained that can be distinguished by a combination of restriction enzymes. Sequence analysis of these clones revealed the presence of three homologous members whose genes, all consisting of three exons and two introns, are almost identical except the exon 2 sequences encoding the inhibitor domain called "WAP motif"; the intron sequences are related to each other with sequence similarities of 93-98%, whereas the exon 2 sequences exhibited only 60-77% similarities among the three members. The extreme divergence in the exon 2 sequences compared to the highly conserved intron sequences may be generated by accelerated mutations confined in a short stretch of the genes following recent duplication events of a single ancestral gene. An RNase protection assay indicated that the messages of the elafin family members are abundantly expressed in the trachea and intestine, suggesting that the most likely selective forces for the accelerated evolution are extrinsic proteinases produced by invasive microorganisms.

1. Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK) is involved in the development of tumour metastasis. Wf-536, (+)-(R)-4-(1-Aminoethyl)-N-(4-pyridyl) benzamide monohydrochloride, a novel inhibitor of ROCK, inhibits tumour metastasis in some animal models. To metastasise, tumour cells have to disturb the tight intercellular junctions and the basement membrane matrix of the host tissue, which, respectively, create an intercellular barrier and the extracellular membrane. To clarify the mechanism of Wf-536 in inhibition of tumour metastasis, we analysed the effect of Wf-536 on the transition of tumour cells through the host cell layer and the basement membrane in in vitro systems. 2. In a coculture system of human fibrosarcoma HT1080 cells plated on a monolayer of human ECV304 cells, Wf-536 (0.3-3 micromol/L) inhibited the paracellular infiltration of tumour cells. 3. Wf-536 (3-30 micromol/L) inhibited the invasion of tumour cells through the reconstituted basement membrane (Matrigel) layer. 4. Wf-536 (10-30 micromol/L) inhibited the migration of tumour cells. At 0.3-3 micromol/L, Wf-536 also restrained hepatocyte growth factor/scatter factor (HGF)-induced increases in paracellular permeability of the ECV304 cell layer. 5. These results suggest that Wf-536 suppresses tumour metastasis by both enhancing the barrier function of host cell layers and inhibiting tumour cell motility at the stage of host tissue penetration by metastatic tumour cells.

The primary and gene structures and tissue distribution of porcine SPAI-2, a protein that belongs to the WAP protein superfamily and has a sodium-potassium ATPase inhibitory activity, were determined by molecular cloning and Northern analysis. A full-length cDNA clone was isolated from a porcine duodenum cDNA library. The cDNA insert encoded a polypeptide of 187 amino acids, which is composed of three domains: a hydrophobic presequence of 21 amino acids, a prosegment of 105 amino acids ending with Asp 126 , and the mature SPAI-2 sequence of 61 amino acids beginning with Pro 127. The prosegment contained 16 repeats of a hexapeptide that is highly homologous to the repetitive sequence found in the transglutaminase domain of the human elafin, an elastase-specific inhibitor that also belongs to the WAP superfamily. The repetitive sequence was demonstrated to be a good substrate of transglutaminase using a recombinant preparation produced in Escherichia coli. A porcine genomic library was then screened for the SPAI gene. Characterization and sequencing of positive clones indicated that the gene is similar to the elafin gene, having 3 exons encoding the 5-untranslated region and signal sequence, pro-SPAI, and 3-untranslated region, respectively. Northern blot analysis revealed intestine-specific expression of SPAI mRNA; the message was especially abundant in the small intestine. ProSPAI was also found in the circulation. The similarity of proSPAI to elafin in the domain structure, the acid-labile nature of the cleavage site (Asp 126 -Pro 127 ), and the fact that the major form of SPAI in the plasma is proSPAI strongly suggest that proSPAI is not the precursor but rather it is the native form of SPAI. Like elafin, therefore, SPAI appears to be a new type of biologically active substance with a transglutaminase substrate domain that acts as an anchoring sequence. SPAI-21 is a 61-amino-acid peptide isolated from porcine duodenum as a Na ϩ ,K ϩ -ATPase inhibitor (1) and is now considered as an inhibitor specific for monovalent cation-transporting ATPases (2); however, whether this inhibitory action observed in vitro is its true physiological function remains to be established since the IC 50 is relatively high (ϳ10 M). It belongs to the WAP protein superfamily that has the "four-disulfide core" (3) or "WAP" motif (4); the members include, among others, mucous proteinase inhibitor MPI (5-7) and elafin, an elastase inhibitor that was isolated by Wiedow et al. (8) from the horny layers of patients with psoriasis. MPI and elafin are well characterized at the molecular level; for example, their domain structures and gene structures have been determined (9, 10). Less is known about the molecular biology of SPAI. We therefore cloned cDNA from a porcine duodenum cDNA library, determined its nucleotide sequence to deduce the primary structure of the precursor, and examined the exon-intron organization of the corresponding gene. The results indicated that SPAI is much more similar to elafin than to MPI in its domain and gene struc...

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