Low-density lipoprotein receptor class A repeats are O-glycosylated in linker regions [Cell Biology]

May 27th, 2014 by Pedersen, N. B., Wang, S., Narimatsu, Y., Yang, Z., Halim, A., Schjoldager, K. T.-B. G., Madsen, T. D., Seidah, N. G., Bennett, E. P., Levery, S. B., Clausen, H.

The low-density lipoprotein receptor (LDLR) is crucial for cholesterol homeostasis and deficiency in LDLR functions cause hypercholesterolemia. LDLR is a type I transmembrane protein that requires O-glycosylation for stable expression at the cell surface. It has previously been suggested that LDLR O-glycosylation is found N-terminal to the juxtamembrane region. Recently we identified O-glycosylation sites in the linker regions between the characteristic LDLR class A repeats in several LDLR-related receptors using the 'SimpleCell' O-glycoproteome shotgun strategy. Herein, we have systematically characterized O-glycosylation sites on recombinant LDLR shed from HEK293 SimpleCells and CHO wild-type cells. We find that the short linker regions between LDLR class A repeats contain an evolutionarily conserved O-glycosylation site at position -1 of the first cysteine residue of most repeats, which in wild-type CHO cells is glycosylated with the typical sialylated core 1 structure. The glycosites in linker regions of LDLR class A repeats are conserved in LDLR from man to Xenopus and found in other homologous receptors. O-glycosylation is controlled by a large family of polypeptide GalNAc-transferases. Probing into which isoform(s) contributed to glycosylation of the linker regions of the LDLR class A repeats by in vitro enzyme assays suggested a major role of GalNAc-T11. This was supported by expression of LDLR in HEK293 cells, where knockout of the GalNAc-T11 isoform resulted in the loss of glycosylation of three out of four linker regions.