Intracellular processing, glycosylation, and cell surface expression of human metapneumovirus attachment glycoprotein.
The biosynthesis and posttranslational processing of human metapneumovirus attachment G glycoprotein were investigated. After pulse-labeling, the G protein accumulated as three species with molecular weights of 45,000, 50,000, and 53,000 (45K, 50K, and 53K, respectively). N-Glycosidase digestion indicated that these forms represent the unglycosylated precursor and N-glycosylated intermediate products, respectively. After an appropriate chase, these three naive forms were further processed to a mature 97K form. The presence of O-linked sugars in mature G protein was confirmed by O-glycanase digestion and lectin-binding assay using Arachis hypogaea (peanut agglutinin), an O-glycan-specific lectin. In addition, in the O-glycosylation-deficient cell line (CHO ldlD cell), the G protein could not be processed to the mature form unless the exogenous Gal and GalNAc were supplemented, which provided added evidence supporting the O-linked glycosylation of G protein. The maturation of G was completely blocked by monensin but was partially sensitive to brefeldin A (BFA), suggesting the O-linked glycosylation of G initiated in the trans-Golgi compartment and terminated in the trans-Golgi network. Enzymatic deglycosylation analysis confirmed that the BFA-G was a partial mature form containing N-linked oligosaccharides and various amounts of O-linked carbohydrate side chains. The expression of G protein at the cell surface could be detected by indirect immunofluorescence staining assay. Furthermore, cell surface immunoprecipitation displayed an efficient intracellular transport of G protein.
Liu
Li
L
Department of Medical Microbiology and Infectious Diseases, the University of Manitoba, Winnipeg, Manitoba, Canada.
Bastien
Nathalie
N
Li
Yan
Y
D000818:Animals; D020126:Brefeldin A; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D019084:Fluorescent Antibody Technique, Indirect; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D029121:Metapneumovirus / Q000235:genetics / Q000378:metabolism*; D008985:Monensin; D011499:Protein Processing, Post-Translational; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D014759:Viral Envelope Proteins; C116204:attachment protein G; D020126:Brefeldin A; D008985:Monensin