O-linked N,N'-diacetyllactosamine (LacdiNAc)-modified glycans in extracellular matrix glycoproteins are specifically phosphorylated at subterminal N-acetylglucosamine

Abstract

The terminal modification of glycans by β4 addition of N-acetylgalactosamine to N-acetylglucosamine with formation of the N,N-diacetyllactosediamine (LacdiNAc) moiety has been well documented for a number of N-linked glycoproteins and peptides, like neurohormones. Much less is known about O-glycoproteins in this regard because only human zona pellucida glycoprotein 3 (ZP3) and bovine proopiomelanocortin were reported to be LacdiNAc-modified. In searching for mammalian proteins modified with O-linked LacdiNAc we identified six positive species among nine endogenous and recombinant O-glycoproteins, which were extracellular matrix, or matrix-related proteins. These are ZP3 and the five novel LacdiNAc-positive species ECM1, AMACO, nidogen-1, α-dystroglycan, and neurofascin. The mass spectrometric analyses revealed a core 2-based tetrasaccharide as the common structural basis of O-linked LacdiNAc that could be further modified, similar to the type 2 LacNAc termini, with fucose, sialic acid, or sulfate. Here, we provide structural evidence for a novel type of mucin-type O-glycans that is strictly specific for LacdiNAc termini: sugar phosphorylation with formation of GalNAcβ1-4(phospho-)GlcNAc. The structural details of the phosphatase-labile compound were elucidated by MS(2) analysis of tetralysine complexes and by MS(n) measurements of the permethylated glycan alditols. Phospho-LacdiNAc was detected in human HEK-293 as well as in mouse myoblast cells and in bovine brain tissue.

FIGURE 1.

MALDI-MS/MS of m/z 1024.5 from permethylated O-glycan alditols from recombinant neurofascin. These data are representative of the MS/MS data of m/z 1024 from all analyzed proteins. All fragment masses are sodium adducts if not indicated otherwise. The formation of the Y-ion m/z 284 indicates the presence of a disubstituted HexNAc-ol originating from a core 2 structure, whereas there is no signal corresponding to a linear core 1 tetrasaccharide with a monosubstituted HexNAc-ol (m/z 298 or 316).

FIGURE 2.

ESI-MS/MS spectrum of the peptide LETASPPTR (molecular mass (M), 971.5 Da) modified with a core 2-based LacdiNAc tetrasaccharide (H₁N₃, M 1742.8 Da) from recombinant dystroglycan (hDG6), expressed in HEK-293 (1) (A) and endogenous α-dystroglycan purified from human muscle (B). Both spectra are based on the doubly charged precursor ion 871.9²⁺, representing the peptide modified with Hex₁HexNAc₃ (771.3 Da). Both spectra show identical fragmentation patterns (with the exception of the fragment at m/z 1539.8) even though they were registered on different mass spectrometers. The fragment at m/z 1539.8 was not recorded in spectrum B due to a mass range restriction at m/z 1400.

FIGURE 3.

Schematic domain topology of LacdiNAc-modified glycoproteins. Only those O-glycosylation sites that were either published previously or identified in the course of this study are shown. The recombinantly expressed and analyzed sequence stretch of the respective protein is marked by a box. Squares, mucin-type O-glycans; L, LacdiNAc-modified O-glycans.

FIGURE 4.

Western blot analysis of recombinant and native O-glycoproteins with a specific anti-LacdiNAc-antibody. 5 μg of each protein was loaded onto the SDS-gel. Due to differences in protein molecular mass and in the degree of O-glycosylation, the staining intensities of individual proteins cannot be regarded as quantitatively comparable. First lane, hDG8; second lane, ECM1; third lane, AMACO-P2; fourth lane, nidogen-1; fifth lane, ZP3-IV; sixth lane, MUC1-S; seventh lane, bovine fetuin. The asterisk marks artificial staining of keratin.

FIGURE 5.

ESI-MS/MS spectrum of the N-terminal tryptic peptide (m/z 1733) from the deletion construct of human α-dystroglycan, hDGdel2 (1). Fragmentation pattern and oxonium ions at m/z 407 (HexNAc₂ + H⁺) and m/z 487 (HexNAc₂p + H⁺) indicate a modification with the phosphorylated LacdiNAc tetrasaccharide (H₁N₃P). M, molecular mass.

FIGURE 6.

Selected ion traces of the LC-chromatogram of tryptic peptides from recombinant α-dystroglycan (hDGdel2) that were either untreated or treated with sulfatase or phosphatase prior to analysis by ESI-MS. The ion trace of the tryptic peptide (SLVPR + HexNAc + H⁺, m/z 774.4 in blue) is shown compared with the phosphoglycan-specific ion trace (m/z 487, HexNAc₂p + H⁺ in red). Glycan composition and peptide identity were confirmed for each chromatographic peak by MS/MS (data not shown).

FIGURE 7.

MS2 spectrum of the tetralysine-phosphoglycan complex K₄/H₁N₃-P (molecular mass, 1402 Da) (K₄, tetralysine). Elimination of phosphate as H₃PO₄ (98 Da) and ligand dissociation into free tetralysine (5304 Da) and the glycan chain indicate a modification with phosphate. Sulfate substitution would be indicated by desulfation of glycan without ligand dissociation from tetralysine. The fragment spectrum reveals a phospho modification of the subterminal HexNAc.

FIGURE 8.

ESI-MSⁿ spectra of permethylated O-glycan alditols from recombinant α-dystroglycan (hDG8). A, MS² spectrum of the permethylated phosphoglycan H₁N₃p + H⁺ (m/z 1050) as a methanol elimination product. The structures of the glycan alditol at m/z 1050.5 and of the fragment at m/z 360.2 are included. The fragment at m/z 360.2 can be interpreted as indicating a 3-phosphoester of the subterminal GlcNAc. B, MS³ spectrum of the B_2α-fragment (N₂P) at m/z 553.5 revealing a terminal HexNAc (no methanol elimination) and the elimination of methaphosphate (79 Da). All fragments are proton adducts.

FIGURE 9.

ESI-MS/MS of precursor ion at m/z 1072. 6 from permethylated bovine brain O-glycans corresponds to the sodium adduct of phosphorylated tetrasaccharide HN₃P (M-32).