Alternative splicing governs sulfation of tyrosine or oligosaccharide on peptidylglycine alpha-amidating monooxygenase.

Peptidylglycine a-amidating monooxygenase (PAM) catalyzes the COOH-terminal a-amidation of neuroendocrine peptides through the sequential action of monooxygenase and lyase domains contained within this bifunctional protein.Alternative splicing leads to the expression of soluble and integral membrane bifunctional PAM proteins as well as a soluble monofunctional monooxygenase.In order to determine how alternative splicing affects post-translational modification of PAM proteins, we investigated the sulfation of PAM proteins expressed in stably transfected hEK-293 cells.Metabolic labeling with [s6S]SOi-or [SsS]methionine and immunoprecipitation demonstrated that [s6S]SO:-was efficiently incorporated into PAM proteins that have the noncatalytic exon A region following the monooxygenase domain (PAM-1 and PAM-4) and into a soluble bifunctional PAM protein (PAM-3).Alkaline hydrolysis, radiosequencing, and deglycosylation experiments demonstrated the presence of a sulfated tyrosine ( T y P ) in the COOH-terminal domain of PAM-3 and multiple sulfated O-glycans in the exon A region of PAM-1 and PAM-4.A mutant PAM-3 protein in which w was changed to Alam (PAM-3/Y965A) was not sulfated and exhibited monooxygenase and lyase activities similar to those of wild type PAM-3.Pulse-chase and temperature block experiments showed that the PAM-3/Y98SA protein exits the trans-Golgi network faster than wild type PAM-3.Thus inclusion of exon A results in the sulfation of O-glycans, while elimination of the transmembrane domain results in the sulfation of w.Production of bioactive peptides in the nervous and endocrine systems is accomplished by post-translational processing of precursor proteins during their passage through the secretory pathway.COOH-terminal a-amidation is a late modification that is often rate-limiting and essential for generating bioactive peptides (1,2).A bifunctional enzyme, peptidylglycine a-amidating monooxygenase (PAM; EC 1.14.17.3),' catalyzes the sequential two-step conversion of glycine-extended peptides into COOH-terminal amidated peptides: peptidylglycine Grants DK-32949 and DA-00098.