Analysis of structural differences between Ly-5 molecules of T- and B-cells.

A group of closely related high mol. wt (Mr) membrane glycoproteins is expressed with varying Mr on different subpopulations of lymphocytes, but the different Mr forms share the Ly-5 and T200 antigenic determinants. The Ly-5 molecule expressed by thymocytes has an Mr of 175,000 (175ly5). The antigenically related molecule on B-cells has an Mr of 210,000 (210ly5). It is not known whether the variations in size are due to differences in the polypeptide chain, post-translational modifications such as glycosylation, or both. In this report we examine the glycosylation of 175ly5 and 210ly5 to determine whether differences in carbohydrate moieties may account for the different Mr of these two Ly-5 species. Pronase digestion and alkaline borohydride treatment of these molecules labeled in the terminal galactose residues revealed that 210ly5 molecules have a more complex oligosaccharide pattern than 175ly5 molecules. While Ly-5 oligosaccharides from a T-cell tumor line were very similar to those of normal thymocytes, the pattern of Ly-5 carbohydrates from a B-cell tumor were somewhat different than those from normal B-cells. This report also presents evidence for O-linked sugars on Ly-5 molecules.

Co-immunoprecipitation of the Ly-5 molecule and an endogenous protease: a proteolytic system requiring a reducing agent and Ca2+1.

Sodium [3H]borohydride- and [35S]methionine-labeled Ly-5 molecules from mouse thymocytes and T lymphoma cells were isolated with specific antibody and Staphylococcus aureus Cowan I (SaCI) strain; after extensive washing of the complexes, elution with Laemmli's reducing buffer (0.05 M Tris [pH 6.8 or 6.0], 4% sodium dodecyl sulfate [SDS], and 2% 2-mercaptoethanol [2-ME]) resulted in partial breakdown of the isolated Ly-5 molecules from a Mr = 175,000 to 150,000. Other proteins present during the elution step showed no evidence of proteolysis. 2-ME and SDS were required for proteolysis; although addition of exogenous Ca2+ during elution was not necessary, both EDTA and EGTA inhibited breakdown of the molecule that could be overcome by excess Ca2+. Of a variety of protease inhibitors and thiol-reactive agents tested, only TAME and oxidized glutathione blocked proteolysis almost completely. SaCI, serum, and contaminating antibodies were ruled out as the source of the proteolytic activity. More stringent preclearing and washing conditions did not eliminate endogenous proteolysis of the Ly-5 molecule. The endogenous proteolytic fragment had a Mr distinct from the tryptic fragment of the Ly-5 molecule. We conclude that the Ly-5 molecule may be autolytic or tightly associated with a distinct cellular protease.