Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions

Abstract

The low-density lipoprotein receptor (LDLR) and related receptors are important for the transport of diverse biomolecules across cell membranes and barriers. Their functions are especially relevant for cholesterol homeostasis and diseases, including neurodegenerative and kidney disorders. Members of the LDLR-related protein family share LDLR class A (LA) repeats providing binding properties for lipoproteins and other biomolecules. We previously demonstrated that short linker regions between these LA repeats contain conserved Oglycan-sites. Moreover, we found that O-glycan modifications at these sites are selectively controlled by the GalNAc-transferase isoform, GalNAc-T11. However, the effects of GalNAc-T11-mediated O-glycosylation on LDLR and related receptors localization and function are unknown. Here, we characterized O-glycosylation of LDLR-related proteins and identified conserved O-glycosylation sites in the LA linker regions of VLDLR, LRP1, and LRP2 (Megalin) from both cell lines and rat organs. Using a panel of geneedited isogenic cell line models, we demonstrate that GalNAc-T11-mediated LDLR and VLDLR O-glycosylation is not required for transport and cell surface expression and stability of these receptors, but markedly enhances LDL and VLDL binding and uptake. Direct ELISA-based binding assays with truncated LDLR constructs revealed that O-glycosylation increased affinity for LDL by approximately 5-fold. The molecular basis for this observation is currently unknown, but these findings open up new avenues for exploring the roles of LDLR-related proteins in disease.

Probing the contribution of individual polypeptide GalNAc-transferase isoforms to the O-glycoproteome by inducible expression in isogenic cell lines

“…Interestingly, Gal-NAc-T2 was recently associated with PAPS transporter 1/SLC35B2, GP73/GOLM1, GOLIM4, and GLG1 as proteins depleted from endocytic vesicles in response to KO of AP-5 adaptor protein complex, suggesting involvement in the late endosome-to-Golgi retrieval process (59). For GalNAc-T11, the early responders identified were LRP1 and LRP2, which is in agreement with our previous findings and supports our hypothesis that O-glycosylation of these receptors serves regulatory functions (24). We recently characterized O-glycosylation of LDLR-related proteins in cell lines and rat organs, identifying LA linker glycosylation in LDLR, VLDLR, LRP1, LRP1B, SorLA, LRP8, and LRP2 (24), but in HEK cells, most of these genes are expressed at very low levels.…”

“…Another important example is GalNAc-T11, originally shown to have substrate specificities very similar to and overlapping with those of GalNAc-T1-T3 in vitro (15) but later identified by us as the only isoform glycosylating a highly conserved O-glycosite in the short linker regions XC 6 XXXTC 1 X present between the ligand-binding LDL class A repeats (LA modules) of all LDLR-related receptors (56). Moreover, these O-glycans and, in particular, their sialic acids potentiate receptor/ligand binding affinity and endocytosis, at least for LDLR and VLDLR (24). Interestingly, the Drosophila ortholog, dGal-NAcT1 or l(2)35a, is essential for embryonal development (15) and appears to have an analogous function, glycosylating LA modules of the fly lipophorin receptors (24).…”

“…transcription rather than through structural defects in the enzyme proteins (32). We chose to study two isoenzymes, Gal-NAc-T2, a major contributor to the O-glycoproteome of all cells and co-regulator of lipid metabolism, and GalNAc-T11, which selectively glycosylates the LDLR-related receptors (24,25,34). Although both enzymes have been shown to have broad substrate specificities by in vitro analyses, we found that gradual induction of either enzyme in isogenic cell lines resulted in exquisite concomitant increase in glycosylation, selectively of their nonredundant substrate sites.…”

“…Moreover, these O-glycans and, in particular, their sialic acids potentiate receptor/ligand binding affinity and endocytosis, at least for LDLR and VLDLR (24). Interestingly, the Drosophila ortholog, dGal-NAcT1 or l(2)35a, is essential for embryonal development (15) and appears to have an analogous function, glycosylating LA modules of the fly lipophorin receptors (24). Human GALNT11 is a GWAS candidate gene associated with chronic kidney decline, and GalNAc-T11 is critical for glycosylation of the linker regions between the LA modules of the endocytic receptor LRP2.…”

“…cro ARTICLE glycopeptide substrates (12,20,21), but more recently, use of isogenic cell lines with knockout (KO) and/or knock-in (KI) of individual GALNT genes coupled with the development of quantitative differential O-glycoproteomics has provided more comprehensive insight into the contributions of individual Gal-NAc-Ts to the O-glycoproteome (22)(23)(24)(25). Perhaps surprisingly, this latter approach has indicated that the nonredundant contributions of GalNAc-T isoenzymes to the O-glycoproteome appear to be minor, and the majority of O-glycosites are redundantly glycosylated among the isoforms present in a cell (22).…”