Kelly P. Kearse

Multiple Dimeric Forms of Human CD69 Result from Differential Addition of N-Glycans to Typical (Asn-X-Ser/Thr) and Atypical (Asn-X-Cys) Glycosylation Motifs

CD69 is expressed on the surface of all hematopoietically derived leukocytes and is suggested to function as a multipurpose cell-surface trigger molecule important in the development and activation of many different cell types. Human CD69 contains only a single consensus sequence for N-linked oligosaccharide addition within its extracellular domain (Asn-Val-Thr), yet exists as two distinct glycoforms that are assembled together into disulfide-linked homodimers and heterodimers. The molecular basis for human CD69 heterogeneity has remained elusive. In the current report we show that human CD69 glycoforms are generated before the egress of CD69 proteins from the endoplasmic reticulum to the Golgi and are synthesized under conditions where Golgi processing is inhibited, effectively ruling out the possibility that CD69 heterogeneity results from the differential processing of a single glycosylation site in the Golgi complex. Importantly, these data demonstrate that contrary to current belief, not one but two sites for N-glycan addition exist within the human CD69 extracellular domain and identify the second, “cryptic” CD69 N-glycan attachment site as the atypical Cys-containing glycosylation motif, Asn-Ala-Cys. The results in this study provide a molecular basis for human CD69 heterogeneity and show that multiple dimeric forms of human CD69 result from the variable addition of N-glycans to atypical and typical glycosylation motifs within the CD69 extracellular domain.

CALNEXIN ASSOCIATION IS NOT SUFFICIENT TO PROTECT T CELL RECEPTOR ALPHA PROTEINS FROM RAPID DEGRADATION IN CD4+CD8+ THYMOCYTES

During T cell development, assembly of the mutisubunit T cell receptor (TCR) complex is regulated by the differential stability of newly synthesized TCRα molecules, having a half-life of approximately 20 min in immature CD4+CD8+ thymocytes compared with >75 min in mature T cells. The molecular basis for TCRα instability in CD4+CD8+ thymocytes is unknown but has been postulated to involve abnormalities in N-glycan processing and calnexin assembly as perturbation of these pathways markedly destabilizes TCRα proteins in all other T cell types examined. Here, we compared the processing of TCRα glycoproteins and their assembly with calnexin and calreticulin chaperones in CD4+CD8+ thymocytes and splenic T cells. These studies show that TCRα glycoproteins synthesized in CD4+CD8+ thymocytes were processed in a similar manner as those made in splenic T cells and that TCRα proteins stably associated with calnexin in both cell types. Interestingly, however, TCRα association with the calnexin-related molecule calreticulin was decreased in CD4+CD8+thymocytes compared with splenic T cells. Finally, TCRα degradation in CD4+CD8+ thymocytes was impaired by inhibitors of proteasome activity, which was correlated with stabilization of calnexin·TCRα complexes. These data demonstrate that calnexin association is not sufficient to protect TCRα proteins from rapid degradation in CD4+CD8+ thymocytes, suggesting that additional components of the quality control system of the endoplasmic reticulum operate to ensure the proper folding of nascent TCRα glycoproteins.

Monoclonal antibody DS6 detects a tumor‐associated sialoglycotope expressed on human serous ovarian carcinomas

A newly developed murine monoclonal antibody, DS6, immunohistochemically reacts with an antigen, CA6, that is expressed by human serous ovarian carcinomas but not by normal ovarian surface epithelium or mesothelium. CA6 has a limited distribution in normal adult tissues and is most characteristically detected in fallopian tube epithelium, inner urothelium and type 2 pneumocytes. Pre‐treatment of tissue sections with either periodic acid or neuraminidase from Vibrio cholerae abolishes immunoreactivity with DS6, indicating that CA6 is a neuraminidase‐sensitive and periodic acid‐sensitive sialic acid glycoconjugate (“sialoglycotope”). SDS‐PAGE of OVCAR5 cell lysates has revealed that the CA6 epitope is expressed on an 80 kDa non–disulfide‐linked glycoprotein containing N‐linked oligosaccharides. Two‐dimensional non‐equilibrium pH gradient gel electrophoresis indicates an isoelectric point of approximately 6.2 to 6.5. Comparison of the immunohistochemical distribution of CA6 in human serous ovarian adenocarcinomas has revealed similarities to that of CA125; however, distinct differences and some complementarity of antigen expression were revealed by double‐label, 2‐color immunohistochemical studies. The DS6‐detected CA6 antigen appears to be distinct from other well‐characterized tumor‐associated antigens, including MUC1, CA125 and the histo‐blood group–related antigens sLea, sLex and sTn. Int. J. Cancer 88:866–872, 2000.

Modification of the T cell antigen receptor (TCR) complex by UDP-glucose:glycoprotein glucosyltransferase. TCR folding is finalized convergent with formation of alpha beta delta epsilon gamma epsilon complexes.

Most T lymphocytes express on their surfaces a multisubunit receptor complex, the T cell antigen receptor (TCR) containing α, β, γ, δ, ε, and ζ molecules, that has been widely studied as a model system for protein quality control. Although the parameters of TCR assembly are relatively well established, little information exists regarding the stage(s) of TCR oligomerization where folding of TCR proteins is completed. Here we evaluated the modification of TCR glycoproteins by the endoplasmic reticulum folding sensor enzyme UDP-glucose:glycoprotein glucosyltransferase (GT) as a unique and sensitive indicator of how TCR subunits assembled into multisubunit complexes are perceived by the endoplasmic reticulum quality control system. These results demonstrate that all TCR subunits containing N-glycans were modified by GT and that TCR proteins were differentially reglucosylated during their assembly with partner TCR chains. Importantly, these data show that GT modification of most TCR subunits persisted until assembly of CD3αβ chains and formation of CD3-associated, disulfide-linked αβ heterodimers. These studies provide a novel evaluation of the folding status of TCR glycoproteins during their assembly into multisubunit complexes and are consistent with the concept that TCR folding is finalized convergent with formation of αβδεγε complexes.

Calnexin associates with monomeric and oligomeric (disulfide-linked) CD3delta proteins in murine T lymphocytes.

The antigen-binding receptor expressed on most T lymphocytes consists of disulfide-linked clonotypic αβ heterodimers noncovalently associated with monomeric CD3γ,δ,ε proteins and disulfide-linked ζζ homodimers, collectively referred to as the T cell antigen receptor (TCR) complex. Here, we examined and compared the disulfide linkage status of newly synthesized TCR proteins in murine CD4+CD8+ thymocytes and splenic T cells. These studies demonstrate that CD3δ proteins exist as both monomeric and oligomeric (disulfide-linked) species that differentially assemble with CD3ε subunits in CD4+CD8+ thymocytes and splenic T cells. Interestingly, unlike previous results on glucose trimming and TCR assembly of CD3δ proteins in splenic T cells (Van Leeuwen, J. E. M., and K. P. Kearse (1996) J. Biol. Chem. 271, 9660–9665), we found that glucose residues were not invariably removed from CD3δ glycoproteins prior to their assembly with CD3ε subunits in CD4+CD8+thymocytes. Finally, these studies show that calnexin associates with both monomeric and disulfide-linked CD3δ proteins in murine T cells. The data in the current report demonstrate that CD3δ proteins exist as both monomeric and disulfide-linked molecules in murine T cells that differentially associate with partner TCR chains in CD4+CD8+ thymocytes and splenic T cells. These results are consistent with the concept that folding and assembly of CD3δ proteins is a function of their oxidation state.

Mutational analysis of conserved amino acids in the T cell receptor α-chain transmembrane region: a critical role of leucine 112 and phenylalanine 127 for assembly and surface expression

Correct assembly of all TCR complex polypeptides is essential for its cell surface expression and function. The transmembrane region of the TCRalpha chain is highly conserved and to gain insight into the structural and functional role of these residues, single amino acid substitutions were introduced and surface expression and signaling ability studied in T hybridoma cells. Introduction of acid residues within the TCRalpha chain transmembrane region were mostly tolerated, indicating that the net charge within this region of the TCR complex is not crucial to either assembly or signaling. However, mutations of leucine 112 or phenylalanine 127 to aspartic acids (L112D or F127D, respectively) resulted in dramatic loss of surface expression and, therefore, their signaling ability. Intracellular flow cytometry showed that the mutant TCRalpha polypeptides were present at levels comparable to wild-type, indicating that the reduced surface expression was not a consequence of impaired protein survival. The defect was characterized by immunoprecipitation and showed that residues L112 and F127 were involved in early interactions with the CD3 complex. A large proportion of the TCRalpha chain mutants L112D and F127D consisted of immature protein, indicative of a problem during early assembly of the TCR. Our findings provide evidence for the involvement of the conserved L112 and F127 residues of the TCRalpha chain transmembrane region in the assembly process of the TCR complex.

Importance of the T cell receptor α-chain transmembrane distal region for assembly with cognate subunits

Antigen recognition by alphabeta T lymphocytes is mediated via the multisubunit TCR complex consisting of invariant CD3gamma,delta,epsilon and zeta chains associated with clonotypic TCRalpha and beta molecules. Charged amino acids located centrally within the TCRalpha transmembrane region are necessary and sufficient for assembly with the CD3deltaepsilon heterodimer. Previously, we have shown that deletion of 6-12 amino acids from the carboxy terminus of the TCRalpha-chain dramatically abrogates surface TCR expression, suggesting that the distal portion of the TCRalpha transmembrane region contains information that regulates the assembly and/or intracellular transport of TCR complexes. We have examined in more detail the molecular basis for reduced TCR expression in T cells bearing truncated TCRalpha chains. We found that in contrast to wild-type (wt), variant TCRalpha proteins missing the last nine C-terminal amino acids did not associate with core CD3gamma,delta,epsilon chains and were not assembled into disulphide-linked alphabeta heterodimers. The stability of newly synthesised wt and variant TCRalpha molecules was similar, showing that the abrogated surface TCR expression was not a consequence of impaired protein survival. Nevertheless, truncated TCRalpha chains still assembled with the chaperon protein calnexin in the endoplasmic reticulum, indicating that the distal portion of the TCRalpha transmembrane region is not essential for calnexin interaction. These data document a role for the distal portion of the TCRalpha transmembrane region in the assembly of TCR complexes and provide a molecular basis for reduced TCR expression in cells bearing truncated TCRalpha chains.

A useful radiolabeling method for detection of T cell receptor glycoproteins bearing immature and mature N-linked glycans

The antigen receptor expressed on most T lymphocytes, the T cell receptor (TCR), is a multisubunit complex containing TCR alpha,beta and CD3-gamma,delta, epsilon and zeta chains, in the form alphabetadeltaepsilongammaepsilonzetazeta. The intracellular transport of TCR proteins is routinely measured by the extent of processing of N-linked glycan chains present on TCR alpha,beta and CD3 gamma,delta subunits, which are converted from immature (high mannose) oligosaccharides in the endoplasmic reticulum (ER) to mature (complex type) species within the Golgi complex. In the current study, a radiolabeling method is described that is useful for the study of TCR glycoproteins bearing immature and mature N-linked oligosaccharide chains. Specifically, we report the use of [3H] galactose as a radioactive tracer of TCR proteins containing glucose and galactose residues, present in immature and mature N-linked oligosaccharides, respectively. This technique is rapid, simple, and sensitive, and provides a unique approach for studying the processing and subcellular localization of T cell receptor proteins.