Identification of a 40-kDa Cell Surface Sialoglycoprotein with the Characteristics of a Major Influenza C Virus Receptor in a Madin-Darby Canine Kidney Cell Line

doi:10.1074/jbc.270.30.17815

Identification of a 40-kDa Cell Surface Sialoglycoprotein with the Characteristics of a Major Influenza C Virus Receptor in a Madin-Darby Canine Kidney Cell Line (*)

From the Institut für Virologie, Philipps-Universitt Marburg, Robert-Koch-Straße 17, D-35037 Marburg, Federal Republic of Germany

^¶ To whom correspondence should be addressed:
Institut fr Virologie, Philipps-Universitt Marburg, Robert-Koch-Str. 17, D-35037 Marburg, Germany.
Tel.: 06421-285360; Fax: 06421-285482.

Abstract

Infection of cells by influenza C virus is known to be initiated by virus attachment to cell surface glycoconjugates containing N-acetyl-9-O-acetylneuraminic acid. Using an in vitro virus binding assay, we have detected this carbohydrate on several glycoproteins of Madin-Darby canine kidney cells (type I), a polarized epithelial cell line permissive for infection with influenza C virus. Among these proteins, only one was found to be present to a significant extent on the cell surface. This protein, gp40, was characterized as an O-glycosylated (mucin-type) integral membrane protein of 40 kDa, which was predominantly localized on the apical plasma membrane of filter-grown cells. It is a major cell surface sialoglycoprotein in this cell line and was shown to be subject to constitutive and rapid endocytosis. Thus, this glycoprotein can mediate not only the binding of influenza C virus to the cell surface, but also its delivery to endosomes, where penetration occurs by membrane fusion. Other highly sialylated cell surface glycoproteins were also detected but did not mediate influenza C virus binding to a significant extent, indicating that only gp40 contains 9-O-acetylated sialic acids.

Footnotes

↵^§ This work was conducted in partial fulfillment of the requirements for the Dr. rer. nat. degree from FB17, Philipps-Universitt Marburg.
↵* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 286. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

Neu5Ac

N-acetylneuraminic acid

BCIP

5-bromo-4-chloro-3-indolyl phosphate 4-toluidine salt

BSA

bovine serum albumine

HAU

hemagglutinating units

NBT

nitro blue tetrazolium chloride

Neu5,9Ac₂

N-acetyl-9-O-acetylneuraminic acid

LCA

Lens culinaris agglutinin

MDCK

Madin-Darby canine kidney

PAGE

polyacrylamide gel electrophoresis

PBS

phosphate-buffered saline

PMSF

phenylmethylsulfonylfluoride

PNA

peanut agglutinin

SNA

Sambucus nigra agglutinin

WGA

wheat germ agglutinin.
- Received April 7, 1995.
- Revision received May 25, 1995.