Identification of mammalian glycoproteins with type-I LacdiNAc structures synthesized by the glycosyltransferase B3GALNT2

Abstract

The type-I LacdiNAc (LDN; GalNAcβ1-3GlcNAc) has rarely been observed in mammalian cells except in the O-glycan of α-dystroglycan, in contrast to type-II LDN structures (GalNAcβ1-4GlcNAc) in N- and O-glycans that are present in many mammalian glycoproteins, such as pituitary and hypothalamic hormones. Although a β1,3-N-acetylgalactosaminyltransferase 2 (B3GALNT2; type-I LDN synthase) has been cloned, the function of type-I LDN in mammalian cells is still unclear, as its carrier protein(s) has not been identified. In this study, using HeLa cells, we demonstrate that inhibition of Golgi-resident glycosyltransferase increases the abundance of B3GALNT2-synthesized type-I LDN structures, recognized by Wisteria floribunda agglutinin (WFA). Using isotope-coded glycosylation site-specific tagging (IGOT)-LC/MS analysis of Lec8 Chinese hamster cells lacking galactosylation and of cells transfected with the B3GALNT2 gene, we identified the glycoproteins that carry B3GALNT2-generated type-I LDN in their N-glycans. Our results further revealed that LDN presence on low-density lipoprotein receptor-related protein 1 and nicastrin depends on B3GALNT2, indicating the occurrence of type-I LDN in vivo in mammalian cells. Our analysis also uncovered that most of the identified glycoproteins localize to intracellular organelles, particularly to the endoplasmic reticulum. Whereas B4GALNT3 and B4GALNT4 synthesized LDN on extracellular glycoproteins, B3GALNT2 primarily transferred LDN to intracellular glycoproteins, thereby clearly delineating proteins that carry type-I or type-II LDNs. Taken together, our results indicate the presence of mammalian glycoproteins carrying type-I LDN on N-glycans and suggest that type-I and type-II LDNs have different roles in vivo.

Keywords: B3GALNT2; Golgi; LC/MS analysis; N-linked glycosylation; WFA; Wisteria floribunda agglutinin (WFA); carbohydrate structure; endoplasmic reticulum; endoplasmic reticulum (ER); glycoprotein; glycosyltransferase; intracellular glycoproteins; type-I LacdiNAc.

Figure 1.

WFA recognizes LacdiNAc synthesized by B3GALNT2. A, lectin blotting (LB, upper panel) or immunoblotting (IB, lower panel) of whole-cell lysates (10 μg) from HeLa+B3GALNT2-GFP cells cultured with DMSO (BFA(−); lanes 1-3) or 15 μg/ml BFA (BFA(+); lanes 4-6) for 2 days. HeLa+B3GALNT2-GFP cells were treated with nontargeting siRNA (siCont; lanes 2 and 5) or B3GALNT2-targeting siRNA (siRNA; lanes 3 and 6) for 2 days before BFA treatment. WFA (left) or WGA (right) were used for LB. An anti-GFP antibody (left) to detect B3GALNT2-GFP or an anti-GAPDH antibody (right) were used for IB. B, WFA-captures (left) or WGA-captured glycoproteins (right) from whole-cell lysates used in A were detected by WFA (left) or WGA (right), respectively. C, WFA-blotting of WFA-captured glycoproteins treated with EndoH (lanes 2, 5, and 8) or PNGaseF (lanes 3, 6, and 9) from HeLa+GFP cells (lanes 1–3) or HeLa+B3GALNT2-GFP cells (lanes 4–9) cultured with DMSO (BFA(−), lanes 1–6) or BFA (BFA(+), lanes 7–9).

Figure 2.

WFA(+) proteins in B3GALNT2-transfected cells. A, lectin blotting of WFA-captured (lanes 1 and 2) or WGA-captured (lanes 5 and 6) glycoproteins or whole-cell lysates (input; lanes 3, 4, 7, and 8) from Lec8 cells (lanes 1, 3, 5, and 7) or B3GALNT2-myc-His-transfected Lec8 cells (lanes 2, 4, 6, and 8). WFA (lanes 1–4) or WGA (lanes 5–8) were used as described in “Experimental procedures” (lectin blot). B, silver staining of WFA-captured glycoproteins (lanes 1 and 2) or whole-cell lysates (input; lanes 3 and 4) from Lec8 cells (lanes 1 and 3) or B3GALNT2-myc-His–transfected Lec8 cells (lanes 2 and 4).

Figure 3.

IGOT-LC/MS analysis of WFA capture proteins. A, comparison of WFA-binding glycoproteins identified by IGOT-LC/MS analysis between Lec8 cells (gray, Lec8, 152) and B3GALNT2-transfected Lec8 cells (black, +B3GALNT2, 306). A total of 140 glycoproteins were in the overlap between the two sets. (The results show combined data on proteins identified in the first and second experiments. Because proteins identified by the Mascot search included a few isoforms, deferent isoforms corresponding to the same gene name were assumed to be one protein). B and C, the identified proteins were classified based on the Cell Part members (B) or Organelle members (C) within the Cellular Component categorized by a bioinformatic protein classification system, PANTHER. The numbers of genes classified from WFA(+)-glycoproteins of Lec8 cells (light gray, WFA(+)_Lec8) or B3GALNT2-transfected Lec8 cells (black, WFA(+)_+B3GALNT2) are shown as solid bars. As controls, the bars from Amide-80-column(+)-proteins of Lec8 cells (white, Amide(+)_Lec8) or B3GALNT2-transfected Lec8 cells (dark gray, Amide(+)_+B3GalNT2) are also indicated.

Figure 4.

A comparison of N-glycan profiles by MALDI-TOF-MS analysis. A and B, MALDI-TOF-MS analysis of N-glycans from Lec8 cells (A, red, Lec8) and B3GALNT2-transfected Lec8 cells (B, green, +B3GALNT2). A peak height of (011)-N-glycan is indicated as a height reference (dotted square, dotted line). Note, increases of HexNAc-containing structures (021, 031, and 041) were remarkable in B3GALNT2-transfected Lec8 cells (red rectangles). The glycan composition of major peaks containing hexose (Hex), N-acetylhexosamines (HexNAc), and deoxyhexose (dHex) is indicated at the top of a peak (Hex, HexNAc, dHex). The numbers were obtained after subtraction of the trimannosyl-chitobiose core structure (Man₃-GlcNAc₂) of N-glycan. a.u.: arbitrary units.

Figure 5.

B3GALNT2 targets intercellular proteins but not secreted proteins. A and B, WFA-blotting of WFA-captured glycoproteins from the cultured medium (A, secreted) or whole-cell lysates (B, intracellular) of HeLa cells transfected with mock (lane 1), B3GALNT2 (lane 2), B4GALNT3 (lane 3), or B4GALNT4 (lane 4) plasmid DNA. HeLa transfectants were treated with DMSO (BFA(−), lanes 1–4) or 15 μg/ml BFA (BFA(+), lanes 5–8) for 2 days after transfection in (B). C, immunoblotting of WFA-captured glycoproteins (lanes 1–6) or whole-cell lysates (lanes 7–12) from untreated HeLa cells (lanes 1 and 7) or HeLa transfectants (lanes 2–6, 8-12). HYOU1, PIGS, ADGRG1, and NCSTN were detected. Amounts of WFA-captured HYOU1 and PIGS increased significantly when B3GALNT2s were transfected into the cells (lanes 2 and 4).

Figure 6.

Up-regulation of LacdiNAc(+)-glycoproteins in B3GALNT2-transfected Lec8 cells. Immunoblotting of WGA-captured (lanes 1 and 2) or WFA-captured (lanes 3 and 4) glycoproteins or whole-cell lysates (lanes 5 and 6) from Lec8 cells (lanes 1, 3, and 5) or B3GALNT2-transfected Lec8 cells (lanes 2, 4, and 6). PIGS, PGAP1, HYOU1, NCSTN, and LRP1 were analyzed.

Figure 7.

Confirmation of LacdiNAc(+)-N-glycans on a candidate protein. A and B, immunoprecipitated NCSTN (A) or immunoprecipitated LRP1 (B) was examined by lectin blotting (LB) with WFA for LacdiNAc or WGA. Data from groups “control IgG” (lanes 1 and 2) and “protein-specific IgG” (lanes 3–6) were compared. N-glycosylation instances were removed by PNGaseF (lanes 5 and 6), and the amounts of target proteins were confirmed by immunoblotting (IB, lanes 3–6).

Figure 8.

The presence of type-I LacdiNAc synthesized by endogenous B3GALNT2. Immunoblotting of WFA-captured glycoproteins (lanes 1–3) or whole-cell lysates (lanes 4–6) from HeLa cells treated with nontargeting siRNA (siCont; lanes 1 and 4) or B3GALNT2-targeting siRNA (siRNA_#1, lanes 2 and 5; siRNA_#1–3, lanes 3 and 6) for 4 days. Amounts of NCSTN and LRP1 bearing WFA(+)-glycans decreased depending on siRNA treatment (lanes 2 and 3) without a decrease in the total amounts of NCSTN and LRP1 (lanes 5 and 6).