Alternative splicing governs sulfation of tyrosine or oligosaccharide on peptidylglycine alpha-amidating monooxygenase.
Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the COOH-terminal alpha-amidation of neuro-endocrine 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 [35S]SO4(2-) or [35S]methionine and immunoprecipitation demonstrated that [35S]SO4(2-) 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 (Tyr965) 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 Tyr965 was changed to Ala965 (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/Y965A 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 Tyr965.
Yun
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HY
Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Keutmann
H T
HT
Eipper
B A
BA
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D006801:Humans; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D009154:Mutation; D009844:Oligosaccharides / Q000378:metabolism*; D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology; D013431:Sulfates / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
D017931:DNA Primers; D009097:Multienzyme Complexes; D009844:Oligosaccharides; D013431:Sulfates; D014443:Tyrosine; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase