A new hydrophobic glass-fiber support is presented, which is well suited to the electrophoretic transfer of proteins from polyacrylamide gels and subsequent protein-chemical analysis. Modified glass-fiber sheets are easily prepared by chemical reaction of the surface with poly(methyl-3,3,3-trifluoropropylsiloxane) in trifluoroacetic acid. The modification is stable during electroblotting, amino acid sequence analysis and hydrolysis. The siliconized glass fiber exhibits a high protein-binding capacity, allows the application of well-established staining procedures, and does not interfere with the analytical methods of modern protein chemistry at the low picomole level. Samples separated by electrophoresis and immobilized on hydrophobic supports fail to exhibit any detectable contamination in amino acid sequence analysis hence allowing the high performance of the available protein-chemical methods to be exploited.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1988.tb14308.x

A new siliconized-glass fiber as support for protein-chemical analysis of electroblotted proteins.

Two-dimensional electrophoretic separation and immobilization of proteins onto inert membranes for subsequent amino acid sequence and amino acid composition analysis is described as a rapid procedure for the identification or characterization of proteins from complex mixtures. This method avoids the drawbacks of classical purification and isolation methods which involve time-consuming operations with low resolution and, often, insufficient yields. Excellent overall yields of minor amounts (in the low microgram range) using this method allow for sequence determination of yet inaccessible proteins. Solubilized cell proteins of mouse brain were separated by high resolution two-dimensional electrophoresis and electroblotted onto a siliconized glass fiber membrane. The immobilized proteins were stained with Coomassie Brilliant Blue R-250, and twelve proteins spots were then submitted to both Edman degradation and amino acid analysis. Proteins were identified by comparison of the experimentally determined amino acid composition with a dataset derived from the Protein Identification Resource (PIR) protein sequence database. Eight out of twelve proteins tested were identified by amino acid analysis and confirmed by N-terminal sequence determination.

Identification of mouse brain proteins after two-dimensional electrophoresis and electroblotting by microsequence analysis and amino acid composition analysis

By comparative analysis of the hemagglutinin-esterase (HE) protein of mouse hepatitis virus strain S (MHV-S) and the HE protein of influenza C virus, we found major differences in substrate specificities. In striking contrast to the influenza C virus enzyme, the MHV-S esterase was unable to release acetate from bovine submandibulary gland mucin. Furthermore, MHV-S could not remove influenza C virus receptors from erythrocytes. Analysis with free sialic acid derivatives revealed that the MHV-S HE protein specifically de-O-acetylates 5-N-acetyl-4-O-acetyl sialic acid (Neu4, 5Ac2) but not 5-N-acetyl-9-O-acetyl sialic acid (Neu5,9Ac2), which is the major substrate for esterases of influenza C virus and bovine coronaviruses. In addition, the MHV-S esterase converted glycosidically bound Neu4,5Ac2 of guinea pig serum glycoproteins to Neu5Ac. By expression of the MHV esterase with recombinant vaccinia virus and incubation with guinea pig serum, we demonstrated that the viral HE possesses sialate-4-O-acetylesterase activity. In addition to observed enzymatic activity, MHV-S exhibited affinity to guinea pig and horse serum glycoproteins. Binding required sialate-4-O-acetyl groups and was abolished by chemical de-O-acetylation. Since Neu4,5Ac2 has not been identified in mice, the nature of potential substrates and/or secondary receptors for MHV-S in the natural host remains to be determined. The esterase of MHV-S is the first example of a viral enzyme with high specificity and affinity toward 4-O-acetylated sialic acids.

The hemagglutinin-esterase of mouse hepatitis virus strain S is a sialate-4-O-acetylesterase.

Publisher Summary Soon after the first isolation of an influenza C virus from a patient, it became obvious that this virus differs from other myxoviruses in several aspects. Pronounced differences have been observed in the interactions between the virus and cell surfaces, suggesting that influenza C virus attaches to the receptors different from those recognized by other myxoviruses. While influenza A and B viruses agglutinate erythrocytes from many species, including humans, the spectrum of erythrocytes agglutinated by influenza C virus is much more restricted. Erythrocytes from rats, mice, and adult chickens are suitable for hemagglutination and hemadsorption tests; cells from other species, however, react not at all or only poorly with influenza C virus. Differences are also observed so far as hemagglutination inhibitors are concerned. A variety of glycoproteins have been shown to prevent influenza A and B viruses from agglutinating erythrocytes. In the case of influenza C virus, rat serum was for a long time the only known hemagglutination inhibitor. A difference in the receptors for influenza C virus and other myxo-viruses was also suggested by studies on the receptor-destroying enzyme. The ability of influenza C virus to inactivate its own receptors was reported soon after the first isolation of this virus from a patient. However, the influenza C enzyme did not affect the receptors of other myxoviruses and, conversely, the receptor-destroying enzyme of either of the latter viruses was unable to inactivate the receptors for influenza C virus on erythrocytes. While the enzyme of influenza A and B virus was characterized as a neuraminidase in the 1950s, even with refined methodology no such activity was detectable with influenza C virus. It is now known that both the receptor-binding and receptor-destroying activities, as well as the fusion activity of influenza C virus are mediated by the only glycoprotein present on the surface of the virus particle. The structure and functions of this protein, which is designated as HEF, are reviewed in this chapter.

/pdf/structure-and-function-of-the-hef-glycoprotein-of-influenza-4k2r3gyv2i.pdf

Structure and function of the HEF glycoprotein of influenza C virus.

Differential fatty acid selection during biosynthetic S-acylation of a transmembrane protein (HEF) and other proteins in insect cells (Sf9) and in mammalian cells (CV1).

Isolation of the influenza C virus glycoprotein in a soluble form by bromelain digestion

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Isolation of the influenza C virus glycoprotein in a soluble form by bromelain digestion