title: Isolation of the measles virus hemagglutinin protein in a soluble form by protease digestion.
abstract: The hemagglutinin (H) glycoprotein was isolated in a soluble form by digesting measles virus particles with an endoproteinase, Asp-N (from a Pseudomonas fragi mutant). Digestion of H with Asp-N brought about glycopeptides in three different forms, depending on the cleaving site: AHD, which has an M(r) of 66,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and which formed a disulfide-linked homodimer with an M(r) of 132,000, and two monomeric digestion products, AHM-1 (with an M(r) of 64,000) and AHM-2 (with an M(r) of 58,000). The susceptibility of the H glycoprotein to the protease depended on the enzyme concentration. AHD was readily formed at a low concentration of Asp-N, while AHM-1 and AHM-2 required higher and even higher protease concentrations, respectively. All of the cleavage products reacted with monoclonal antibodies to various epitopes of the H protein; however, only AHD showed a significant hemagglutinin activity on African green monkey erythrocytes. The hemagglutinin activities of AHM-1 and AHM-2 were restored after a monoclonal antibody lacking the hemagglutination-inhibiting activity was added to the reaction mixture. AHDs purified by size-exclusion high-pressure liquid chromatography had two associating forms; one had an M(r) higher than and the other an M(r) as high as that of a tetramer. The former was associated noncovalently in addition to having two intermolecular disulfide bonds, and the latter was associated covalently with a single intermolecular disulfide bond and was also duplicated through a noncovalent association. In addition, both AHM-1 and AHM-2, having no intermolecular disulfide bond, were in a dimer form. These results suggest that AHM-1 and AHM-2 are monovalent in the hemagglutinin activity, while AHDs are divalent. Comparative analyses of the N termini of these soluble glycopeptides with the sequence of H suggested that the cysteine residue at position 139 was responsible for the intermolecular disulfide bonding between the monomeric H glycoproteins. The cysteine at position 154 was also suggested to participate in the forming of the intermolecular disulfide bond.
authors.affiliation: Department of Virus Disease and Vaccine Control, National Institute of Health, Tokyo, Japan.
authors.forename: T A
authors.initials: TA
authors.lastname: Sato
authors.forename: M
authors.initials: M
authors.lastname: Enami
authors.forename: T
authors.initials: T
authors.lastname: Kohama
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002522:Chlorocebus aethiops; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D006389:Hemagglutinins, Viral / Q000302:isolation & purification* / Q000378:metabolism; D008459:Measles virus; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D012995:Solubility; D014709:Vero Cells
chemical_list: D006389:Hemagglutinins, Viral; C032397:hemagglutinin protein G, measles virus; D010450:Endopeptidases; D008666:Metalloendopeptidases; C058921:endoproteinase Asp-N