Analysis of the carbohydrate composition of the pancreatic plasmalemmal glycoprotein affinity labeled by short probes for the cholecystokinin receptor

J Biol Chem. 1987 Oct 5;262(28):13850-6.

Abstract

Affinity labeling of the rat pancreatic cholecystokinin (CCK) receptor with decapeptide probes has identified an Mr = 85,000-95,000 protein, distinct from the Mr = 80,000 component previously labeled with 125I-Bolton Hunter-CCK-33. We have characterized the carbohydrate composition of this novel protein labeled with 125I-D-Tyr-Gly-[(Nle28,31)-CCK-26-33] and disuccinimidyl suberate by using chemical and enzymatic deglycosylation and lectin chromatography. The Mr = 85,000-95,000 component was demonstrated to be an N-linked sialoglycoprotein based on neuraminidase digestion to Mr = 75,000-85,000 and endo-beta-N-acetylglucosaminidase F (Endo F) digestion to Mr = 42,000. This was distinct from the Mr = 65,000 product of Endo F digestion of the protein labeled with 125I-Bolton Hunter-CCK-33. Lack of an effect of endo-beta-N-acetylglucosaminidase H demonstrated the absence of N-linked simple oligosaccharides, while products of chemical deglycosylation with hydrogen fluoride and endo-alpha-N-acetylgalactosaminidase supported the absence of O-linked carbohydrate. The presence of at least four oligosaccharide chains on the core protein was suggested by Endo F digestion of the Mr = 85,000-95,000 protein using limiting enzyme conditions. This glycoprotein was retained on wheat germ agglutininagarose and eluted by N,N',N"-triacetylchitotriose. Identification of the Mr = 85,000-95,000 component on the ectodomain of the plasmalemma of intact pancreatic acini confirmed this to be the fully processed form of the CCK-binding protein.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Affinity Labels / metabolism*
  • Animals
  • Carbohydrates / analysis
  • Cell Membrane / metabolism
  • Cholecystokinin / metabolism*
  • Glycoproteins / isolation & purification
  • Glycoproteins / metabolism*
  • Glycoside Hydrolases
  • Male
  • Molecular Weight
  • Pancreas / metabolism*
  • Peptide Fragments*
  • Rats
  • Rats, Inbred Strains
  • Receptors, Cholecystokinin / isolation & purification
  • Receptors, Cholecystokinin / metabolism*
  • Sincalide / analogs & derivatives*
  • Sincalide / chemical synthesis
  • Sincalide / metabolism

Substances

  • Affinity Labels
  • Carbohydrates
  • Glycoproteins
  • Peptide Fragments
  • Receptors, Cholecystokinin
  • cholecystokinin (26-33), I-Tyr-Gly-Nle(28,31)-
  • Cholecystokinin
  • Glycoside Hydrolases
  • Sincalide