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Glycosylation of Parathyroid Hormone-Related Peptide Secreted by Human Epidermal Keratinocytes^*

Divisions of Endocrinology and Dermatology, West Haven Veterans Affairs Medical Center West Haven, Connecticut 06516 Yale University School of Medicine New Haven, Connecticut 06510

Address all correspondence and requests for reprints to: Andrew F. Stewart, M.D., Research/151, West Haven Veterans Administration Medical Center, 950 Campbell Avenue, West Haven, Connecticut 06516.

While the gene and mRNA transcripts encoding PTH-related peptide (PTHrP) have been well characterized, the actual secretory form(s) of the peptide is unknown. Accordingly, synthetic and recombinant PTHrPs employed to date for biological and immunological characterization have necessarily been of arbitrary lengths. No prior evidence for glycosylation of PTHrPs has been described. To define the naturally occurring form(s) of this peptide secreted by human epidermal keratinocytes, we have affinity purified, using an anti-PTHrP-(l–36) antibody column, human PTHrP secreted under conditions of protease protection. Human keratinocyte-conditioned medium collected without measures to protect against proteolytic degradation contains multiple PTHrP immunoreactive and bioactive species. In contrast, under conditions of protease protection, human keratinocyte-conditioned medium contains a single 18,000 mol wt (Mr) form of the peptide. In contrast to recombinant and synthetic PTHrPs, which migrate as distinct, well focussed bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this 18,000 Mr PTHrP displays the broad electrophoretic profile of a glycoprotein. Treatment of this peptide with trifluoromethanesulfonic acid, an agent that deglycosylates both O- and N-linked saccharides from their core proteins, shifted the Mr of the protein to approximately 10,000. In contrast, exposure of recombinant PTHrP-(l–141) to the same agent results in no change in electrophoretic mobility. These studies indicate that the 18,000 Mr species of PTHrP secreted by human epidermal keratinocytes is a glycoprotein.

^* This work was supported by the Department of Veterans Affairs (West Haven, CT) and NIH Grants AR-37594 and DK-07276–13.

Received February 4, 1991.

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