The Golgi sialoglycoprotein MG160, expressed in Pichia pastoris, does not require complex carbohydrates and sialic acid for secretion and basic fibroblast growth factor binding.
MG160, a type I membrane sialoglycoprotein of the medial cisternae of the rat Golgi apparatus, shows high homology (over 90%) with CFR, a fibroblast growth factor receptor, and ESL-1, an E-selectin ligand of the cell surface of murine myeloid cells. When Chinese Hamster Ovary (CHO) cells were stably transfected with a cDNA lacking the transmembrane and C-terminus cytoplasmic domain of MG160 (delta TMCT), a fully processed protein of 160 kDa apparent molecular mass was recovered in the culture medium. When these cells were treated with tunicymycin, a 130- to 140-kDa protein was immunoprecipitated from the culture medium. A construct lacking the signal sequence, the single transmembrane, and the cytoplasmic domains of MG160 (delta TMCT-) was integrated at the HIS Pichia pastoris genome site using the expression vector pPIC 9 which possesses a yeast compatible signal sequence (Invitrogen). Recombinant protein accumulated in the medium to approximately 10 mg/L. The yeast recombinant protein lacked complex carbohydrates and sialic acid but bound 125I bFGF. Similarly, rat MG160 subjected to deglycosylation by peptide:N-glycosidase F (PNGase) bound 125I bFGF.
Chen
Y J
YJ
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
Gonatas
N K
NK
D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D001216:Asparagine / Q000378:metabolism; D016466:CHO Cells; D050260:Carbohydrate Metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D003470:Culture Media / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism*; D015870:Gene Expression; D006031:Glycosylation / Q000187:drug effects; D006056:Golgi Apparatus / Q000737:chemistry; D008565:Membrane Proteins / Q000378:metabolism; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D009439:Neuraminidase / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010843:Pichia / Q000235:genetics*; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics; D012795:Sialoglycoproteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000235:genetics; D014415:Tunicamycin / Q000494:pharmacology
D003470:Culture Media; D008565:Membrane Proteins; D011994:Recombinant Proteins; D012795:Sialoglycoproteins; D016222:Fibroblast Growth Factor 2; D014415:Tunicamycin; D001216:Asparagine; D009439:Neuraminidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D019158:N-Acetylneuraminic Acid