Glycosylation of parathyroid hormone-related peptide secreted by human epidermal keratinocytes.
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-(1-36) 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-(1-141) 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.
Division of Endocrinology, West Haven Veterans Affairs Medical Center, Connecticut 06516.
T L
TL
Wu
N E
NE
Soifer
W J
WJ
Burtis
L M
LM
Milstone
A F
AF
Stewart
D015153:Blotting, Western; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004817:Epidermis; D006031:Glycosylation; D006801:Humans; D007231:Infant, Newborn; D015603:Keratinocytes / Q000502:physiology*; D008297:Male; D044162:Parathyroid Hormone-Related Protein; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
C470121:PTHLH protein, human; D044162:Parathyroid Hormone-Related Protein; D011506:Proteins; D011994:Recombinant Proteins