The dimensions of the T lymphocyte glycoprotein leukosialin and identification of linear protein epitopes that can be modified by glycosylation.

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.

A mechanism for inhibition of E-cadherin-mediated cell-cell adhesion by the membrane-associated mucin episialin/MUC1.

“…As a consequence, 20 amino acids in the episialin mucin domain will span about 4 nm, assuming that the globular non-mucin part of the ectodomain, just upstream of the transmembrane domain, does not significantly contribute to the length of the molecule. This corresponds to the length of 20 amino acids, spanning approximately 5 nm, in the mucin domain of leukosialin (CD43), another membrane-associated mucin (Jentoft, 1990;Cyster et al, 1991). It is tempting to speculate that adhesion is no longer affected if the length of episialin is reduced to a size close to that of the cell to substrate distance that is in the range of 10-35 nm (for a review, see Springer, 1990).…”

“…Based on the assumptions mentioned above, the episialin ectodomain containing three repeats, which did not inhibit cell-cell interactions, will span about 50 nm, which is still considerably longer than the distance between two interacting cells. Strikingly, leukosialin extends only 45 nm above the cell surface but can still inhibit cell-cell interactions (Cyster et al, 1991;Ardman et al, 1992;Manjunath et al, 1993). This discrepancy might be explained by the relatively low density of 0-linked glycans on human episialin compared with other membrane-associated mucins, resulting in a more flexible molecule that is relatively less capable of inhibiting adhesion.…”

Phenotype of Rat Monocytes During Acute Kidney Allograft Rejection: Increased Expression of NKR‐P1 and Reduction of CD43

“…Although W3/13 has been shown to bind to a protein epitope [9], the accessibility or conformation of this epitope may be altered by glycosylation. It is well known that W3/13 only reacts with certain, but not all, CD43 isoforms [9]. Nevertheless, we suppose that the large cell size and special phenotype of the weakly W3/13-positive monocytes in our study indicate that either reduced expression or an alteration of the leucosialin molecule is associated with activation or differentiation of the cells.…”

Mycobacterium tuberculosis employs Cpn60.2 as an adhesin that binds CD43 on the macrophage surface

“…CD43 (leukosialin, sialophorin) is a sialylated mammalian glycoprotein that is found on most haematopoietic cells (Carlsson and Fukuda, 1986), including murine MF (Keller et al, 1989), and is notable for its comparatively long extension away from the leukocyte surface (~45 nm) (Cyster et al, 1991). CD43 has been proposed to have a duality of roles with respect to mediating intercellular interactions via both pro-adhesive and anti-adhesive mechanisms (Ostberg et al, 1998).…”