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

Abstract

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.CD69 is a member of the NK gene complex family of type II oligomeric signal transmitting receptor proteins that contain C-type lectin-binding domains and is expressed on a variety of hematopoietically derived cells, including bone marrow cells, monocytes, platelets, T and B lymphocytes, and natural killer cells (1-6). In all cell types examined, CD69 cross-linking transduces intracellular signals that generate a variety of cellular responses, suggesting that CD69 is a pleiotropic immune regulator important in the biology of many different hematopoietic cell types (2, 7-9). A specific ligand for CD69 has not been identified but has been postulated to involve carbohydrate moieties (10, 11).CD69 is encoded by a single nonpolymorphic gene and is expressed in both the mouse and human as disulfide-linked homodimers and heterodimers of differentially glycosylated CD69 polypeptides or glycoforms (12-15). For mouse CD69, three putative N-glycan addition sites (Asn-X-Ser/Thr) exist within the extracellular region, accounting for the capacity to synthesize multiple CD69 glycoforms (3). Human CD69 synthesis is somewhat enigmatic in that only one N-glycan addition sequence (Asn-Val-Thr) has been identified within the extracellular domain (1, 13, 14). The molecular basis for human CD69 heterogeneity is unknown but has been proposed to result from qualititative heterogeneity in chain glycosylation, indicative of immature and mature oligosaccharides on CD69 species (2, 7). Recent studies by Hamann et al. (14) argue against this idea, however, as CD69 polypeptides synt...

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