Not all tissues that express epidermal growth factor (EGF) mRNA and proEGF have the ability to produce mature EGF, an important local, luminal, and circulating growth factor for the gastrointestinal tract. We have observed that although EGF mRNA is expressed in substantial quantities in the rat submandibular gland at the time of birth, mature EGF peptide can be detected only around the time of sexual maturation. We hypothesize that one or more hormonally-regulated specific arginyl esteropeptidase(s), expressed exclusively in the submandibular gland at the time of sexual maturation, are responsible for the post-translational processing of proEGF to its mature peptide form. Tissue-specific expression of kallikrein genes therefore may account for the unique ability of submandibular gland to process proEGF intracellularly. However, nearly all the kallikrein cDNAs so far identified from the mouse and rat species have been shown to express in the submandibular gland. Based on the criteria of tissue co-localization, developmental expression and hormonal responsiveness, we have focused on several kallikrein enzymes to determine which candidates are responsible for EGF processing in rat submandibular gland. We are undertaking the gene transfer and expression approaches to establish our experimental models. First, we have expressed proEGF in mammalian cell lines that produce unprocessed proEGF and subsequently a specific kallikrein enzyme can be introduced and the ensuing pattern of proEGF processing analyzed. The proEGF processing enzymes will be identified by the successful production of mature EGF in the cultured cells. Second, we have expressed individual rat kallikrein enzymes in bacteria E. coli to large quantities and the biochemical evidence of in vitro proEGF processing by kallikreins will be determined by their binding and catalytic activity to synthetic peptides or fusion proteins of proEGF substrate. By determining which kallikrein enzymes are responsible for the intracellular processing of proEGF, we will gain understanding on how regulation of EGF and its processing enzyme genes are coordinated that lead to the production of mature EGF under various developmental, hormonal and pathophysiological conditions.
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