MOLECULAR CHARACTERIZATION AND HORMONE-DEPENDENT EXPRESSION OF THE PORCINE WHEY ACIDIC PROTEIN GENE

A 17.5 kDa protein was isolated from porcine whey by reverse phase HPL C and identified as a putative whey acidic protein (WAP) homologue by sequencing 35 and 40 amino acid residues of the amino- and carboxy-ter minus respectively. Degenerate oligonucleotides to both of these amino acid sequences were designed and used in reverse transcriptase PCR wi th RNA from lactating porcine mammary gland asa template. A 162 bp PCR fragment was detected and sequenced. Compilation of the deduced and d etermined amino acid sequence revealed a protein of 111 amino acids, w hich had approximately 75, 50, 40 and 35% similarity at amino acid lev el to camel, rabbit, rat and mouse WAP respectively. It also included the four-disulphide core characteristic of all WAP proteins and most K unitz-type protease inhibitors. This provides the first unequivocal ev idence for WAP secretion in the pig. SDS PAGE analysis of the whey fra ction showed that WAP is secreted as a major protein in sow's milk fro m farrowing to weaning. The molecular mass of WAP in SDS PAGE was sign ificantly greater than the 11.7 kDa determined from amino acid sequenc e, indicating that porcine WAP is possibly glycosylated. Northern anal ysis detected a single mRNA transcript of approximately 600 bp in porc ine RNA from the mammary gland of lactating sows. To examine the hormo ne-regulated expression of the WAP gene the mammary glands of sows at day 90 of pregnancy were biopsied and explants cultured for 3 days in the presence of various combinations of porcine insulin (I), cortisol (F) and porcine prolactin (P). Northern analysis of RNA extracted from the tissue indicated that WAP gene expression was barely detectable i n the mammary gland prior to culture and there was no increment in exp lants cultured in the presence of I and F. However, a significant incr ease in the accumulation of WAP mRNA was observed in explants cultured in I, F and P. A similar result was observed for beta-casein and alph a-lactalbumin gene expression.