Isolation of immature and mature T cell receptor complexes by lectin affinity chromatography.
The antigen-specific T cell receptor (TCR) is a multisubunit complex composed of at least six different polypeptide chains (alpha, beta, gamma, delta, epsilon, zeta), several of which are glycoproteins. Assembly of the TCR occurs in the endoplasmic reticulum (ER) and involves intermediary complexes of CD3-gamma, delta, epsilon proteins and TCR alpha and -beta molecules. Egress of TCR from the ER and transport through the Golgi apparatus is most often monitored by the sensitivity of TCR glycoproteins to endoglycosidase H (Endo H), an enzyme specific for immature oligosaccharides which have not yet been processed by Golgi glycosidases and glycosyltransferases. Because they are not glycosylated, the subcellular localization of CD3-epsilon and TCR zeta chains cannot be directly determined by Endo H treatment, and therefore must be inferred by association with glycoprotein members of the TCR complex. Thus, when both immature and mature TCR glycoproteins are present within a given sample, this becomes extremely difficult. In this report, we describe a method for the physical separation of immature and mature murine TCR complexes based on processing of N-linked carbohydrate side chains. Specifically, we report the use of wheat germ agglutinin-affinity matrices to separate TCR complexes which have reached the trans Golgi compartment of the cell from those that have not. This technique is rapid, sensitive, and does not affect the integrity of assembled TCR complexes.
Kearse
K P
KP
Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Singer
A
A
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D006056:Golgi Apparatus; D006596:Hexosaminidases / Q000494:pharmacology; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D051379:Mice; D011948:Receptors, Antigen, T-Cell / Q000096:biosynthesis* / Q000187:drug effects / Q000302:isolation & purification*; D014909:Wheat Germ Agglutinins
D011948:Receptors, Antigen, T-Cell; D014909:Wheat Germ Agglutinins; D006596:Hexosaminidases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase