Analysis of human skin mast cell proteins by two-dimensional gel electrophoresis. Identification of tryptase as a sialylated glycoprotein.
Proteins of mast cells purified from human foreskin were separated by 2-D polyacrylamide gel electrophoresis using either nonequilibrium pH gradient electrophoresis or isoelectric focusing in the first dimension and SDS-PAGE in the second dimension. Silver staining showed that a major feature of skin mast cell 2-D protein maps was a variety of relatively abundant proteins in the m.w. range of 29 to 37 kDa and covering a broad pH range from 5.0 to 8.5. Tryptase was identified on Western blots of 2-D-separated proteins by its binding of mAb and of 3H-diisopropylfluorophosphate. The precise distribution of tryptase varied among individuals but this protein generally occupied a continuum of molecular weights between 28 and 37 kDa and ranged in isoelectric point between 5.0 and 6.5. Tryptase was one of a number of mast cell proteins that bound the lectin concanavalin A as well as lectins specific for sialic acid, demonstrating that this enzyme is a sialylated glycoprotein. The diffuse m.w. distribution of skin mast cell tryptase (31 to 36 kDa) observed after SDS-PAGE was reduced to a single band of 30 kDa after treatment with protein-N-glycosidase F to remove asparagine-linked oligosaccharides. This finding suggests that intrinsic m.w. heterogeneity of tryptase in skin mast cells is largely a result of the addition of variable amounts of oligosaccharide to the tryptase polypeptide.
Benyon
R C
RC
Department of Medicine, University of Alberta, Edmonton, Canada.
Enciso
J A
JA
Befus
A D
AD
D002675:Child, Preschool; D053818:Chymases; D015180:Electrophoresis, Gel, Two-Dimensional; D006023:Glycoproteins / Q000032:analysis*; D006031:Glycosylation; D006801:Humans; D008407:Mast Cells / Q000737:chemistry*; D008970:Molecular Weight; D019158:N-Acetylneuraminic Acid; D012697:Serine Endopeptidases / Q000032:analysis*; D012794:Sialic Acids / Q000032:analysis; D012867:Skin / Q000737:chemistry; D053802:Tryptases
D006023:Glycoproteins; D012794:Sialic Acids; D012697:Serine Endopeptidases; C083294:chymase 2; D053818:Chymases; D053802:Tryptases; D019158:N-Acetylneuraminic Acid