Characterization of ERp29, a novel secretion factor of endoplasmic reticulum

Abstract: ERp29 is a ubiquitously expressed endoplasmic reticulum protein strongly conserved in mammalian species. The N-terminal domain of ERp29 is similar to the thioredoxin domain of protein disulfide isomerase (PDI) lacking however the double-cysteine active site motif. The C-terminal domain is a novel five-helical fold. It was hypothesized that ERp29 may function as a molecular chaperone facilitating folding of the secretory proteins in the ER. The current investigation extended our knowledge of ERp29 providing the most up-to-date account of the genetic, molecular and functional features of this protein. Gene structure and expression. Characterization and phylogenetic analysis of the rat, murine and human ERp29 genes demonstrated their common origin and close ortholog relationships. Such characteristics of the 5´ flank as CpG island, the absence of TATA-box, multiple transcription start sites in combination with Sp1-dependent basal transcription and ubiquitous gene expression indicate that ERp29 belongs to the group of constitutively expressed housekeeping genes. Exclusive expression of ERp29 in multicellular organisms in concert with its high expression in the specialized secretory tissues suggests a hypothetical secretory role for ERp29. Functional activity of ERp29. ERp29 is induced upon the proliferation and differentiation of thyroid epithelial cells. Co-immunoprecipitation experiments, sucrose density gradient fractionation of the thyrocytes and affinity chromatography using immobilized Tg demonstrated ATP-independent association of ERp29 with thyroglobulin (Tg), the main secretory protein of thyroid cells, and ER chaperones (BiP and GRp94). Strong association of ERp29 with misfolded Tg and existence of the ER heterocomplexes including ERp29, Tg and other ER chaperones was shown also in the human and rat thyroid glands expressing mutant, transport-incompetent Tg. Surprisingly, despite the presence of the ERretrieval signal, ERp29 is secreted synchronously with Tg. Overexpression of ERp29 significantly increases secretion of Tg whereas inhibition of ERp29 by RNAi gene silencing had an opposite effect, which strongly suggests the essential secretory function of ERp29 in the ER. Substrate-binding sites of ERp29. Mutational analysis revealed two potential peptidebinding sites on the ERp29 surface. First, the highly mobile interdomain linker including a unique Cys157 was shown to be important for the functional activity of ERp29. Second functional site is located in the N-terminal domain and as judged by the analysis of the electrostatic surface is an uncharged cleft that might accommodate proteins of sufficiently large size. ERp29 is a potential target of the unfolded protein response (UPR). We have investigated the potential involvement of ERp29 in the development of UPR. Activation of UPR pathways was demonstrated in the differentiating thyroid epithelial cells and in the endoplasmic reticulum storage diseases (ERSD) caused by the missense mutations in the Tg gene. In both cases we observed induction of ERp29 along with major ER chaperones suggesting that ERp29 is a potential UPR target gene. In conclusion, our study describes ERp29 as a novel type of an ER auxiliary folding/secretory factor that facilitates transport of thyroglobulin and potentially of other secretory proteins to the cell exterior.