The Dimensions of the T Lymphocyte Glycoprotein Leukosialin and Identification of Linear Protein Epitopes That Can Be Modified by Glycosylation

EMBO J. 1991 Apr;10(4):893-902.

Abstract

Leukosialin (CD43) is a major glycoprotein of T lymphocytes whose extracellular domain of 224 amino acids contains on average one O-linked carbohydrate unit per three amino acids. This suggests an unfolded structure for the extracellular domain which has now been established to extend to a length of 45 nm by transmission electron microscopy following low angle rotary shadowing. The antigenicity of rat leukosialin has been studied using nine monoclonal antibodies (MAbs) whose binding is differentially affected by the cell type on which leukosialin is expressed and by the removal of sialic acid. From these observations it appears that the epitopes are affected by glycosylation, yet seven of the nine MAbs reacted clearly with the extracellular domain of leukosialian expressed in an unglycosylated form in Escherichia coli. The MAbs showing this positive reaction included three of the four antibodies whose epitopes were affected by neuraminidase treatment of leukosialin. It thus appears that linear protein epitopes are recognized and that some of these can be modified in the native structure by glycosylation. The positions of the antigenic determinants have been mapped by expressing fusion proteins of different lengths and the identity of one epitope was proven by the binding of two MAbs to an octapeptide expressed as a fusion protein. For three MAbs, the location of epitopes in the native protein was confirmed by electron microscopy of shadowed leukosialin--Fab complexes. Overall it is concluded that leukosialin is a major component at the periphery of the T lymphocyte and that despite its high level of glycosylation, protein determinants are exposed that could be ligands in cell interactions.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies, Monoclonal
  • Antigens, CD*
  • Cell Line
  • Cloning, Molecular
  • Epitopes / analysis*
  • Escherichia coli / genetics
  • Glycosylation
  • Humans
  • Leukosialin
  • Microscopy, Electron
  • Molecular Sequence Data
  • Polymerase Chain Reaction
  • Protein Conformation
  • Rats
  • Recombinant Proteins / immunology
  • Recombinant Proteins / ultrastructure
  • Sequence Homology, Nucleic Acid
  • Sialoglycoproteins / genetics
  • Sialoglycoproteins / immunology
  • Sialoglycoproteins / ultrastructure*
  • T-Lymphocytes / metabolism*
  • Transfection

Substances

  • Antibodies, Monoclonal
  • Antigens, CD
  • Epitopes
  • Leukosialin
  • Recombinant Proteins
  • Sialoglycoproteins
  • Spn protein, rat
  • UN1 sialoglycoprotein, human