Folding and oligomerization properties of a soluble and secreted form of the paramyxovirus hemagglutinin-neuraminidase glycoprotein.
The paramyxovirus SV5 hemagglutinin-neuraminidase (HN) glycoprotein (a type II integral membrane protein) was converted into a soluble and secreted form (HN-F) by replacing the HN signal/anchor domain with a hydrophobic domain that can act as a cleavable signal sequence. Approximately 40% of the HN-F synthesized was secreted from cells (t1/2 approximately 2.5-3 hr). The extracellular HN-F molecules were identified as disulfide-linked dimers and the majority of the population of molecules were resistant to endoglycosidase H digestion. Examination of the oligomeric form of the secreted HN-F, by sucrose density gradient sedimentation, indicated that under conditions where HN was a tetramer, HN-F was found to be a dimer, and no extracellular HN-F monomeric species could be detected. Secreted HN-F was fully reactive with conformation-specific monoclonal antibodies and was enzymatically active as shown by HN-F having neuraminidase activity. Examination of the intracellular HN-F species indicated that HN-F monomers were slowly converted to the disulfide-linked form and that under the sucrose density gradient sedimentation conditions used the HN-F monomers aggregated. Some of the HN-F monomers were degraded intracellularly. These data are discussed in relationship to the seemingly different folding and oligomerization requirements for the intracellular transport of soluble and membrane bound forms of a glycoprotein. The soluble and biologically active form of HN may be suitable for further structural and enzymatic studies.
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Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208-3500.
Lamb
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D000911:Antibodies, Monoclonal / Q000276:immunology; D001692:Biological Transport; D002462:Cell Membrane / Q000201:enzymology / Q000276:immunology; D002478:Cells, Cultured; D004220:Disulfides / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D015707:HN Protein / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology; D007700:Kinetics; D010252:Paramyxoviridae / Q000201:enzymology*; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000737:chemistry; D012995:Solubility
D000911:Antibodies, Monoclonal; D004220:Disulfides; D015707:HN Protein; D021382:Protein Sorting Signals