Interaction of Immobilized Recombinant Mouse C-type Macrophage Lectin With Glycopeptides and Oligosaccharides

Biochemistry. 1994 Jul 5;33(26):8159-66. doi: 10.1021/bi00192a021.

Abstract

Inflammatory and tumoricidal macrophages express galactose- and N-acetylgalactosamine-specific Ca(2+)-dependent lectins on their surfaces. This lectin is a family member of membrane-bound C-type animal lectins and consists of 304 amino acid residues (molecular weight 34,595). In the present study, expression vectors containing a nucleotide sequence corresponding to the carbohydrate-binding domain of mouse macrophage lectin cDNA have been prepared. The carbohydrate-binding specificity of the recombinant macrophage lectin expressed in Escherichia coli was investigated by comparing elution profiles of various glycopeptides having defined carbohydrate structures on immobilized lectins. When elution profiles of high mannose-type and complex-type Asn-linked carbohydrate chains were compared, the degree of retardation from immobilized macrophage lectin column was in the order tetraantennary complex-type with terminal galactosyl residues > triantennary complex-type with terminal galactosyl residues > biantennary complex-type with terminal galactosyl residues > high mannose-type glycopeptides. N-Terminal octapeptides from human glycophorin A that bore three NeuAc alpha 2-3Gal beta 1-3(NeuAc alpha 2-6)GalNAc serine/threonine-linked tetrasaccharide chains and their sequentially deglycosylated derivatives were also applied to this column. Glycopeptides carrying three constitutive GalNAc-Ser/Thr(Tn-antigen) had the strongest affinity, whereas those with fully sialylated carbohydrate tetrasaccharide chains showed weak interaction. The association kinetics of Asn-linked glycopeptides from bovine asialofetuin to recombinant macrophage lectin was determined by surface plasmon resonance spectroscopy. The results indicate k(assoc) value of 1.63 x 10(4) M-1 s-1. The calculated value for Ka was 6.20 x 10(7) M.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Asialoglycoproteins / metabolism
  • Base Sequence
  • Biosensing Techniques
  • Calcium / metabolism
  • Carbohydrate Sequence
  • Chromatography, Affinity
  • Escherichia coli / genetics
  • Fetuins
  • Glycopeptides / metabolism*
  • Lectins / genetics
  • Lectins / metabolism*
  • Macrophages / chemistry*
  • Mice
  • Molecular Sequence Data
  • Oligosaccharides / metabolism*
  • Recombinant Proteins / metabolism
  • Solubility
  • alpha-Fetoproteins / metabolism

Substances

  • Asialoglycoproteins
  • Fetuins
  • Glycopeptides
  • Lectins
  • Oligosaccharides
  • Recombinant Proteins
  • alpha-Fetoproteins
  • asialofetuin
  • Calcium