Studies on the carbohydrate moiety and on the biosynthesis of rat C-reactive protein.
Rat C-reactive protein (CRP) is a pentameric glycoprotein composed of five apparently identical monomers, two of which form a disulfide-linked dimer (Rasosouli, M., Sambasivam, H., Azadi, P., Dell, A., Morris, H. R., Nagpurkar, A., Mookerjea, S., and Murray, R. K. (1992) J. Biol. Chem. 267, 2947-2954). In this study, the nature of the oligosaccharide chain of rat CRP was investigated by fast atom bombardment-mass spectrometry (FAB-MS), and general features of its biosynthetic pathway were also analyzed. FAB-MS, electrospray-mass spectrometry, and linkage analysis demonstrated that each monomer of rat CRP contained one oligosaccharide chain, predominantly a disialylated biantennary structure, attached to Asn-128. The biosynthesis of rat CRP was studied by immunoprecipitation of CRP synthesized in vitro and by cultured hepatocytes. The results revealed that each monomer of rat CRP was synthesized individually as a single-chain precursor with a cleavable signal sequence. The translocated species was sensitive to digestion by endoglycosidase H (endo H), indicating that it possessed a high mannose oligosaccharide. Rat CRP acquired the ability to bind to phosphorylcholine-Sepharose and to form the dimeric and oligomeric species prior to acquiring resistance to endo H. Studies using tunicamycin revealed that the N-linked oligosaccharide present in rat CRP was not required for formation of its dimeric component, oligomerization, ability to bind to phosphorylcholine, or secretion. The non-glycosylated rat CRP, however, was still able to bind to phosphorylcholine-Sepharose and to be secreted by hepatocytes.
Sambasivam
H
H
Department of Biochemistry, University of Toronto, Ontario, Canada.
Rassouli
M
M
Murray
R K
RK
Nagpurkar
A
A
Mookerjea
S
S
Azadi
P
P
Dell
A
A
Morris
H R
HR
D000818:Animals; D001216:Asparagine; D002097:C-Reactive Protein / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002474:Cell-Free System; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000096:biosynthesis* / Q000737:chemistry*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000138:chemical synthesis / Q000302:isolation & purification; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D051381:Rats; D012156:Reticulocytes / Q000378:metabolism; D012794:Sialic Acids / Q000032:analysis; D016339:Spectrometry, Mass, Fast Atom Bombardment
D006023:Glycoproteins; D046911:Macromolecular Substances; D009844:Oligosaccharides; D012794:Sialic Acids; D001216:Asparagine; D002097:C-Reactive Protein