CD2 2d glycans, in contrast to the high mannose dl glycans, are complex. Their disposition is such that they fall on either side of CD2, potentially forming a scaffold into which a second CD2 molecule can slot. An earlier observation that neuraminidase treatment of intact T cells enhances the expression of the anti-CD2R mAb D66 is consistent with the idea that removal of negative charges on one or both glycan(s) may more readily permit CD2 clustering. Hence, the level of sialylation of the CD2 2d glycans mayfinely regulate the clustering process. We have found that exposure of T cells to vibrio cholerae neuraminidase (40 mU/m.L) for 45 min at 37 'C results in a 2-3 fold increase in CM[R expression after a 45 min exposure to enzyme without a change in CD2 copy number as judged by anti-T I I reactivity. In view of the distance of d2 glycans from theCD2R epitope recognized by anti-T113, alteration in mAb reactivity cannot simply be secondary to removal of steric constraint imposed by the glycan on mAb binding. Taken together, these results show that the number of CD2R+ conformers is up-regulated by removal of sialic acid residues on one or both CD2 N-linked glycan(s). Considerable evidence exists to support the notion that carbohydrate moieties on cell surface glycoproteins are altered during physiologic T cell activation. In collaboration with the MS Resource, we plan to profile and quantity N-linked glycans before and after activation. This requires native T cells and the best efforts at sensitivity. The analyses involve electroblotting of the CD2 to PVDF, deglycosylating directly and passing the supernatant through a short SePak to retain the protein. The glycans are not retained; these are collected, methylated and profiled. We have not yet achieved requisite sensitivity to undertake this task, although the recovery is improving.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR010888-03
Application #
6123282
Study Section
Project Start
1998-07-01
Project End
1999-06-30
Budget Start
Budget End
Support Year
3
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Boston University
Department
Type
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118
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