The biochemical characterization of E2, a T cell surface molecule involved in rosettes.
We have previously identified a molecule on the T cell surface, which, in addition to CD2 is involved in the rosette phenomenon. This is a 32-kDa single polypeptide chain which we have termed E2. The studies reported here show striking patterns on the glycosylation status of E2. It is a heavily sialylated and glycosylated molecule, the sugar moieties accounting for almost half of its relative molecular mass (Mr). It carries no N-linked sugar residues, only O-linked oligosaccharides. Despite heavy glycosylation, the molecule appears to behave homogeneously on gel electrophoresis, both in terms of Mr and pI. Neuraminidase treatment of E2 lowered its Mr to 28 kDa; this was further decreased to 18 kDa after removal of O-linked sugar residues by treatment with O-glycanase. An identical reduction in size was observed after treatment with trifluoromethane sulfonic acid, showing that the molecule carries no detectable N-linked sugar residues. Moreover, endoglycosidase F and endoglycosidase H treatment of either the immunoprecipitates from 125I surface-labeled thymocytes, or of a purified preparation of E2, did not reduce the Mr of E2, nor did tunicamycin treatment of T cells. Two-dimensional gel electrophoresis revealed two discrete spots of acidic pI (4.4 and 4.6) that were still seen after neuraminidase treatment, though they had moderately shifted. Pulse-chase experiments revealed a single 28-kDa precursor form that could have been the unsialylated molecule. Finally, sequencing 14 amino acid residues of the N-terminal side revealed no homology with known proteins. Since the sugar moieties of adhesion protein could play an important role, the results obtained in this study will prove valuable to our understanding of the role exerted by the E2 molecule.
Aubrit
F
F
Laboratoire d'Immunologie des Tumeurs de l'Enfant, Institut Gustave-Roussy, Villejuif, France.
Gelin
C
C
Pham
D
D
Raynal
B
B
Bernard
A
A
D000595:Amino Acid Sequence; D000945:Antigens, Differentiation, T-Lymphocyte / Q000032:analysis* / Q000302:isolation & purification; D015180:Electrophoresis, Gel, Two-Dimensional; D006031:Glycosylation; D006801:Humans; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011498:Protein Precursors / Q000032:analysis; D012795:Sialoglycoproteins / Q000032:analysis; D013601:T-Lymphocytes / Q000276:immunology*
D000945:Antigens, Differentiation, T-Lymphocyte; D011498:Protein Precursors; D012795:Sialoglycoproteins