Low density lipoprotein receptor class A repeats are O-glycosylated in linker regions

Abstract

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 knock-out of the GalNAc-T11 isoform resulted in the loss of glycosylation of three of four linker regions.

Keywords: GALNT; Glycosylation; Glycosyltransferase; Lectin; Lipoprotein Receptor-related Protein (LPR); Low Density Lipoprotein (LDL); O-Glycan; Zinc Finger Nuclease.

FIGURE 1.

Analysis of purified shed LDLR. A, SDS-PAGE Western blot analysis of LDLR expressed in HEK293 SC. Equal amounts of purified shed LDLR were digested for 4 h or overnight (ON) with α-GalNAcase and/or PNGase F, and subjected to SDS-PAGE Western blot analysis with anti-LDLR antibody (ab30532) (left panels) and VVA (right panels). Arrows indicate approximate predicted mass shifts. B, graphic depiction of LDLR with illustration of the domain structure and summary of glycosylation sites. Shown above the depicted LDLR are O-glycosites identified in the class A repeat region of LDLR expressed and shed from HEK293 SC. The distribution of unique peptides with defined, partially defined, and unmodified glycosites (naked peptides) is indicated. O-Glycosites identified in the C-terminal stem region of LDLR are indicated immediately below the depiction. In the study by Davis et al. (15) amino acids 721–768 (⁷²¹TQET…VTMS⁷⁶⁸) in the stem region were deleted, which includes all O-glycosites identified in this region. O-Glycosites identified previously through the shotgun approach in 12 human cancer SC lines (19) are shown below the LDLR depiction in the line designated SC glycoproteome. Further below this are the core1 O-glycosites identified here with LDLR expressed and shed from CHO WT cells. The five potential N-glycosylation sites are marked with a fork and amino acid position. HEK, HEK293 SC and CHO, CHO WT, green +, confirmed N-glycan site; red –, site covered by unmodified peptide; nc, site not covered in the analysis.

FIGURE 2.

LC-MS spectra demonstrating HexNAc occupancy on Thr⁶⁷ of LDLR from HEK293 SC but not from HEK293 SC T11-KO. A, CID-MS2 spectrum of HexNAc-occupied O-glycopeptide from HEK293 SC with unspecified O-glycosite. B, ETD-MS2 spectrum of the same peptide from HEK293 SC with the site specified. C, HCD-MS2 spectrum of unoccupied peptide from the same analysis of HEK293 SC. D, HCD-MS2 spectrum of unoccupied peptide from the same analysis derived from HEK293SC T11-KO. White rectangle, HexNAc.

FIGURE 3.

Graphic depiction of conservation of glycosites in the class A repeat region of LDLR. O- and N-glycosites identified in human (Hu) LDLR are designated as described in the legend to Fig. 1 and conserved Thr residues in mouse (Mo), hamster (Ha), and X. laevis (Xe) LDLR are shown in red with a square to illustrate potential O-glycosites. Note also that the putative N-glycosylation site in repeat 2 (Asn⁹⁷, sequence not shown) and the N-glycosite in the linker between repeats 6–7 (Asn²⁷²) are conserved.

FIGURE 4.

O-Glycan attachment sites in the ligand-binding domains of LDLR family proteins. Yellow squares indicate O-GalNAc sites found in SimpleCells (19) between the ligand-binding LDLR class A repeats (gray ovals) including the sites presented in Fig. 1. The majority of O-glycosites are found in the identified sequence motif XX-C₆XXXTC₁-XX. Linker sequences with observed O-glycosites that do not match the sequence motif are marked with a red line. White squares indicate the predicted O-glycosites based on the identified linker sequence motif.

FIGURE 5.

In vitro GalNAc-T enzyme assays with 20-mer peptide substrates covering the linker sequences between class A repeats of LDLR. The rate of incorporation into peptides after 4 h incubation was semi-quantified with degree of incorporation (no GalNAc incorporation (−), <5% ((+)), 5–20% (+), 20–50% (++), 50–80% (+++), and >80% (++++) GalNAc incorporated). Identified acceptor sites in the linker sequences are underlined in bold red and potential acceptor sites in red. ND, not determined.

FIGURE 6.

In vitro GalNAc-T glycosylation of an E. coli-expressed reporter encoding four class A repeats (1–4) of LDLR. The purified reporter construct was incubated with recombinant GalNAc-Ts in glycosylation reactions overnight followed by SDS-PAGE Western blot analysis to probe incorporation of GalNAc by reactivity with VVA. Control staining with anti-HIS was included to evaluate protein load and potential degradation. Ctrl, reaction without GalNAc-T.

FIGURE 7.

Graphic depiction of O-glycosites in LDLR isolated from HEK293 SC without GALNT11. LDLR shed from HEK293 SC with zinc finger nuclease-targeted knock-out of GALNT11 (SC T11-KO) was purified and analyzed by MS as described under “Experimental Procedures.” Mass spectral sequence coverage and O-glycosites identified are shown in lower part (LDLR from SC T11-KO) with comparison to the analysis of LDLR isolated from HEK293 SC and in our shotgun approach also presented in Fig. 1. Designations are as described in the legend to Fig. 1.