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 leuk ocytes and is suggested to function as a multipurpose cell-surface tri gger molecule important in the development and activation of many diff erent 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 assembl ed together into disulfide-linked homodimers and heterodimers. The mol ecular basis for human CD69 heterogeneity has remained elusive, In the current report we show that human CD69 glycoforms are generated befor e the egress of CD69 proteins from the endoplasmic reticulum to the Go lgi and are synthesized under conditions where Gels processing is inhi bited, effectively ruling out the possibility that CD69 heterogeneity results from the differential processing of a single glycosylation sit e in the Golgi complex, Importantly, these data demonstrate that contr ary to current belief, not one but two sites for N-glycan addition exi st within the human CD69 extracellular do main and identify the second , ''cryptic'' CD69 N-glycan attachment site as the atypical Cys-contai ning glycosylation motif, Asn-Ala-Cys, The results in this study provi de a molecular basis for human CD69 heterogeneity and show that multip le dimeric forms of human CD69 result from the variable addition of N- glycans to atypical and typical glycosylation motifs within the CD69 e xtracellular domain.