Michael A. Recny

Structure of the glycosylated adhesion domain of human T lymphocyte glycoprotein CD2

BACKGROUND CD2, a T-cell specific surface glycoprotein, is critically important for mediating adherence of T cells to antigen-presenting cells or target cells. Domain 1 of human CD2 is responsible for cell adhesion, binding to CD58 (LFA-3) expressed on the cell to which the T cell binds. Human CD2 domain 1 requires N-linked carbohydrate to maintain its native conformation and ability to bind CD58. In contrast, rat CD2 does not require N-linked carbohydrate, and binds to a different ligand, CD48. RESULTS The three-dimensional structure of the glycosylated form of domain 1 of human CD2 has been determined by NMR spectroscopy. The overall structure resembles the typical beta-barrel of an immunoglobulin variable domain. Nuclear Overhauser enhancement contacts between the protein and the N-linked glycan have been tentatively identified. CONCLUSION Based on our results, we propose a model showing how the N-linked glycan might be positioned in the human CD2 domain 1 structure. The model provides an explanation for the observed instability of deglycosylated human CD2, and allows residues that are important for CD58 binding to be differentiated from those affecting conformational stability via interactions with the glycan.

GLYCOFORM, GLYCOTYPE LINKAGE AND BRANCHING DETAIL OF THE CD2 ADHESION DOMAIN BY DESOLVATION MASS SPECTROMETRY: SFCI-MS AND ESI-MS

Publisher Summary This chapter discusses the utilization of two recently developed techniques in mass spectrometry for N-glycan characterization. In contrast to the ballistic-ionization approaches utilized in desorption mass spectrometry, the current state-of-the-art technology employs direct liquid injection of the solvent and dissolved analytes using various techniques of aerosol formation. The unifying principles are (1) the spatial dispersion of the solvated analyte by generating an aerosol from the bulk liquid and (2) the subsequent desolvation, and hence isolation in the gas phase of single molecules of the analyte from the microscopic solvent/solute clusters. Three mass spectrometry (MS) ion sources are dependent on the principles of desolvation and aerosol formation; thermal spray-MS, electrospray ionization-MS (ESI-MS), and supercritical fluid/chemical ionization (SFCI-MS). Of the techniques currently available, ESI-MS and SFCI-MS probably represent the least energetic or the softest molecular ionization approach that also encompasses remarkable sensitivity. These two techniques can be used independently to characterize separate components of carbohydrate structure. ESI-MS, in a more non-evasive manner, can be used to profile glycan glycoform distribution from isolated glycopeptides, while SFCI-MS is most effective in characterizing the details of carbohydrate structure such as linkage, branching, and primary sequence. Using this latter approach, glycoproteins are first deglycosylated and labeled at the reducing-end, chemically modified, and derivatized.