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.
Highlights
The type-I LacdiNAc (LDN; GalNAc1–3GlcNAc) has rarely been observed in mammalian cells except in the O-glycan of ␣-dystroglycan, in contrast to type-II LDN structures (GalNAc1– 4GlcNAc) in N- and O-glycans that are present in many mammalian glycoproteins, such as pituitary and hypothalamic hormones
To detect LDN glycans, Wisteria floribunda agglutinin (WFA) was used as reported previously [12]. Because it was not clear whether other lectins recognize type-I LDN better than WFA does, we initially employed lectin microarray analysis of whole-cell lysates prepared from HeLa cells and HeLa cells stably expressing B3GALNT2
In addition to WFA, Bauhinia purpurea lectin and Solanum tuberosum lectin recognized the glycoproteins from B3GALNT2-transfected cells, better than those from HeLa cells, suggesting that these three lectins can bind to type-I LDN glycans
Summary
To detect LDN glycans, WFA was used as reported previously [12]. Because it was not clear whether other lectins recognize type-I LDN better than WFA does, we initially employed lectin microarray analysis of whole-cell lysates prepared from HeLa cells and HeLa cells stably expressing B3GALNT2. The peak of (031) was subjected to tandem MS (MS/MS) analysis, detecting the fragment ion with m/z 527.25 corresponding to the LDN fragment (data not shown) These results revealed that the WFA-captured glycopeptides contained type-I LDN glycans, which were synthesized and up-regulated upon B3GALNT2 overexpression, suggesting that the identified glycoproteins could be carrier molecules of type-I LDN in Lec cells. The WFA and WGA signals were eliminated by PNGaseF treatment (Fig. 7, A and B, lanes 5 and 6), indicating that the glycan synthesized by the expressed B3GALNT2 was present on N-glycans of these glycoproteins These results confirmed that the identified glycoproteins carried type-I LDN glycans synthesized by B3GALNT2 on N-glycans in HeLa and Lec cells. We propose that type-I and type-II LDNs (which are present on different glycoproteins) have distinct functions
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