The vasoactive intestinal peptide receptor on intact human colonic adenocarcinoma cells (HT29-D4). Evidence for its glycoprotein nature

Biochem J. 1987 Feb 15;242(1):185-91. doi: 10.1042/bj2420185.

Abstract

We have previously shown that the mono [125I]iodinated vasoactive intestinal peptide (125I-VIP) could be covalently cross-linked on intact colonic adenocarcinoma cells (HT29). A major Mr 67,000 and a minor Mr 120,000 cross-linked polypeptides have been characterized [Muller, Luis, Fantini, Abadie, Giannellini, Marvaldi & Pichon (1985) Eur. J. Biochem. 151, 411-417]. The glycoprotein nature of these species was investigated using endo-beta-acetylglucosaminidase F (Endo F) treatment, enzymic and chemical desialylation and wheat germ agglutinin (WGA)-Sepharose affinity chromatography. Affinity-labelled VIP-binding proteins solubilized by Nonidet P-40 bound to WGA-Sepharose and could be eluted specifically with N-acetyl-D-glucosamine. Treatment with Endo F resulted in an increased electrophoretic mobility of both polypeptides. The major and the minor VIP-binding proteins were converted respectively into Mr 47,000 and 100,000 species, indicating removal of 20 kDa of N-linked oligosaccharides. Deglycosylation with trifluoromethanesulphonic acid also led to a 20 kDa loss in mass of the Mr 67,000 component, indicating the absence of additional O-linked sugars on this polypeptide. The presence of sialic acid on the major VIP-binding protein was demonstrated after treatment of intact cells with neuraminidase or by chemical desialylation with hydrochloric acid. We conclude from this study that the VIP receptor from intact HT29-D4 cells is a glycoprotein with N-linked oligosaccharide side chains containing sialic acid.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylglucosaminidase
  • Adenocarcinoma / analysis
  • Carrier Proteins / analysis
  • Cell Line
  • Chromatography, Affinity
  • Colonic Neoplasms / analysis
  • Glycoproteins / analysis*
  • Humans
  • Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase
  • Receptors, Gastrointestinal Hormone*
  • Receptors, Vasoactive Intestinal Peptide
  • Sialic Acids / analysis
  • Vasoactive Intestinal Peptide / metabolism

Substances

  • Carrier Proteins
  • Glycoproteins
  • Receptors, Gastrointestinal Hormone
  • Receptors, Vasoactive Intestinal Peptide
  • Sialic Acids
  • Vasoactive Intestinal Peptide
  • Acetylglucosaminidase
  • Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase