N– linked glycosylation modulates dimerization of protein disulfide isomerase family A member 2 ( PDIA 2)

Protein disulfide isomerase ( PDI ) family members are important enzymes for the correct folding and maturation of proteins that transit or reside in the endoplasmic reticulum ( ER ). The human PDI family comprises at least 19 members that differ in cell type expression, substrate specificity and post‐translational modifications. PDI family  A member 2 ( PDIA 2, previously known as PDI p) has a similar domain structure to prototypical PDI (also known as PDIA 1), but the function and post‐translational modifications of PDIA 2 remain poorly understood. Unlike most PDI family members, PDIA 2 contains three predicted N ‐linked glycosylation sites. By site‐directed mutagenesis and enzymatic deglycosylation, we show here that all three Asn residues within the potential N ‐linked glycosylation sites of human PDIA 2 (N127, N284 and N516) are glycosylated in human cells. Furthermore, mutation of N284 to glycosylation‐null Gln increases formation of a highly stable disulfide‐bonded PDIA 2 dimer. Nevertheless, in H e L a cells, both wild‐type and N 127/284/516 Q mutant PDIA 2 proteins localize to the ER , but not the ER –Golgi intermediate compartment, suggesting that glycosylation is important for PDIA 2 protein–protein interactions but not subcellular localization. Finally, we identified human major histocompatibility complex class 1 antigens ( HLA ‐ A , B , C ) as potential binding partners of PDIA 2, suggesting an involvement for PDIA 2 in antigen presentation in addition to its previously described roles in autoimmunity and Parkinson's disease. These results further characterize this poorly defined member of the PDI family. Structured digital abstract •  Calreticulin  and  PDIA2   colocalize by fluorescence microscopy ( View interaction ) •  PDIA2  and  PDIA1   colocalize  by  fluorescence microscopy  ( View interaction )