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. 1995 Jan;69(1):513–516. doi: 10.1128/jvi.69.1.513-516.1995

Isolation of the measles virus hemagglutinin protein in a soluble form by protease digestion.

T A Sato 1, M Enami 1, T Kohama 1
PMCID: PMC188601  PMID: 7983748

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.

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Selected References

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