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. 2024 Sep;300(9):107628.
doi: 10.1016/j.jbc.2024.107628. Epub 2024 Aug 2.

In vivo mapping of the mouse Galnt3-specific O-glycoproteome

Kruti Dalal  1 , Weiming Yang  1 , E Tian  2 , Aliona Chernish  1 , Peggy McCluggage  1 , Alexander J Lara  1 , Kelly G Ten Hagen  2 , Lawrence A Tabak  3
Affiliations

In vivo mapping of the mouse Galnt3-specific O-glycoproteome

Kruti Dalal et al. J Biol Chem. 2024 Sep.

Abstract

The UDP-N-acetylgalactosamine polypeptide:N-acetylgalactosaminyltransferase (GalNAc-T) family of enzymes initiates O-linked glycosylation by catalyzing the addition of the first GalNAc sugar to serine or threonine on proteins destined to be membrane-bound or secreted. Defects in individual isoforms of the GalNAc-T family can lead to certain congenital disorders of glycosylation (CDG). The polypeptide N-acetylgalactosaminyltransferase 3 (GALNT)3-CDG, is caused by mutations in GALNT3, resulting in hyperphosphatemic familial tumoral calcinosis due to impaired glycosylation of the phosphate-regulating hormone fibroblast growth factor 23 (FGF23) within osteocytes of the bone. Patients with hyperphosphatemia present altered bone density, abnormal tooth structure, and calcified masses throughout the body. It is therefore important to identify all potential substrates of GalNAc-T3 throughout the body to understand the complex disease phenotypes. Here, we compared the Galnt3-/- mouse model, which partially phenocopies GALNT3-CDG, with WT mice and used a multicomponent approach using chemoenzymatic conditions, a product-dependent method constructed using EThcD triggered scans in a mass spectrometry workflow, quantitative O-glycoproteomics, and global proteomics to identify 663 Galnt3-specific O-glycosites from 269 glycoproteins across multiple tissues. Consistent with the mouse and human phenotypes, functional networks of glycoproteins that contain GalNAc-T3-specific O-glycosites involved in skeletal morphology, mineral level maintenance, and hemostasis were identified. This library of in vivo GalNAc-T3-specific substrate proteins and O-glycosites will serve as a valuable resource to understand the functional implications of O-glycosylation and to unravel the underlying causes of complex human GALNT3-CDG phenotypes.

Keywords: GalNAc-T; Galnt; O-GalNAc; O-glycosylation; glycopeptides; glycosyltransferase; hyperphosphatemic familial tumoral calcinosis (HFTC); mass spectrometry.

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Conflict of interest statement

Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1
Scheme of workflow. A, the tissues were dissected from C57BL/6NHsd (WT) and Galnt3-null (Galnt3−/−) mice. A heatmap of the relative abundance of GalNAc-T3 in tissues reported from https://www.proteomicsdb.org/ is shown. Due to the high abundance of GalNAc-T3 in both SMGs and SLGs and well-established sexual differences in SMGs in rodents, SMG and SLG from male and female mice were dissected and analyzed separately. B, O-glycopeptides and peptides were extracted from tissues using the extraction of O-glycopeptides (ExoO) method. C, TMTpro16-plex, labeled purified O-glycopeptides and peptides from each tissues derived from WT and Galnt3−/− mice. D, labeled samples were combined into 24 fractions using high-pH reversed-phase liquid chromatography. E, data were acquired on LC-MS/MS with HCD-pd-EThcD. F, O-glycoproteomics data were analyzed using MSFragger, pGlyco3.0, and O-pair search with Metamorpheus and proteomics data, and reporter ion intensity of TMT tags was analyzed using PD 2.5. GALNT, polypeptide N-acetylgalactosaminyltransferase 3; HCD, higher-energy collisional dissociation; SLG, sublingual gland; SMG, submandibular gland; TMT, tandem mass tag.
Figure 2
Figure 2
Expression of proteins in Galnt3−/−versus WT tissues. A, heatmap, unsupervised hierarchical clustering, of relative abundance of GalNAc-Ts in different tissues reported from https://www.proteomicsdb.org/. B, heatmap showing protein abundance of GalNAc-T2 and GalNAc-T11 across different WT tissues from our quantitative proteomics data. C, protein abundance of GalNAcT-2,3,4,5,7,10,12 from our proteomics data for male and female salivary glands. D, bar graphs show the percentage of proteins with altered abundance levels in Galnt3−/− tissues relative to WT. E, protein abundance ratios of Galnt3−/−/WT across tissues, are represented as volcano plots. Each dot represents a protein. Gray, blue, and red dots represent unchanged, decreased (foldchange <1.5) and increased (foldchange >1.5) protein abundance, respectively, in the absence of Galnt3. GALNT, polypeptide N-acetylgalactosaminyltransferase 3.
Figure 3
Figure 3
Mapping of O-glycosites from different tissues. A, Galnt3−/− effects on O-glycosite abundance across tissues, represented as volcano plots. Each dot represents a glycosite. Gray, blue, and red dots represent unchanged, decreased (foldchange <1.5), and increased (foldchange >1.5) O-glycosite abundance, respectively. B–E, euler diagrams of glycopeptide sequences, glycopeptides, glycoproteins, and glycan compositions separately identified by MSFragger-Glyco, pGlyco3.0, and O-pair. The area proportional Euler diagram represents sets and their relationships, illustrating the common and distinct O-glycopeptides/peptides/glycans identified using MSFragger, O-pair, and pGlyco3.0. GALNT, polypeptide N-acetylgalactosaminyltransferase 3.
Figure 4
Figure 4
Catalog of GalNAcT-3 specific O-glycosites across tissues.A, the bar graph shows the percentage of unchanged and changed O-glycosites in tissues dissected from Galnt3−/−mice. B, glycoprotein abundance across tissues, represented as volcano plots. Each dot represents a glycoprotein that corresponds to GalNAc-T3-specific O-glycosites. Gray, blue, and red dots represent unchanged, decreased (foldchange <1.5), and increased (foldchange >1.5) glycoprotein abundance, respectively. C, dot plot of GalNAc-T3 isoform specific O-glycosites identified in more than three tissue types. Each blue dot represents a GalNAc-T3 substrate. The x-axis represents the tissue types and the y-axis represents the GalNAc-T3 substrate (UniProtAccession_O-glycosite position in the protein). Q8BI84_T1070∗ glycosite was identified at T1086; T1102 and Q6PZE0_T1389∗ had multiple sites at T1552; T1715; T1878; T2041; T2204; T2367; T2530; T2693; T2856; T3019; T3182; T3345; T3508; T3671; T3834; T3997; T4160; T4323; T4486; T4649; T4812; T4975; T5138; T5301; T5464; T5627; T5790; T5953; T6116; T6279; T6442; T6605; T6768; T6931; and T7094. GALNT, polypeptide N-acetylgalactosaminyltransferase 3.
Figure 5
Figure 5
Mapping of O-glycosites in SMGs and SLG. A, heatmap of O-glycosites identified in SMGs and SLGs from both sexes from Galnt3−/−versus WT. B, Venn diagram of GalNac-T3 substrates identified in the salivary glands based on sex. C, heatmap of GalNAc-T3 specific sites identified in the transporters category. D, In vitro glycosylation workflow to validate osteocalcin (Bglap) S57 as a GalNAc-T3-specific substrate. E, MS2 spectrum of S57 site on recombinant mouse osteocalcin (Bglap) glycosylated by murine GalNAc-T3 and GalNAc (13C6) followed by IMPa cleavage. GALNT, polypeptide N-acetylgalactosaminyltransferase 3; IMPa, immunomodulating metalloprotease; SMG, submandibular gland; SLG, sublingual gland.
Figure 6
Figure 6
Analysis of glycoproteins corresponding to GalNAc-T3 substrates in biological pathways. (A) GO term analysis, (B) KEGG pathway analysis, (C) mammalian phenotype (MP) analysis of glycoproteins identified as GalNAc-T3 substrates. GE, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.
Figure 7
Figure 7
STRING network of glycoproteins identified as GalNAc-T3 substrates. Shown are all proteins identified as GalNAcT-3 substrates across all tissues examined. Lines indicate the strength and confidence of the data support. The arrow indicates the GalNAc-T3 protein which was included in the analysis. Extracellular matrix (ECM) remodeling and proteins (highlighted in red nodes) including laminins (lama2, lama3, and lama3), collagens (col18a1 and col4a2), proteoglycan core proteins (Vcan, Acan, Bcan, and Ncan) appear to a hub for protein-protein interactions in skeletal morphology pathway. Several proteins such as Vwf, F12, Serpinc1, Kng1, APP, Klk1, and Klk1b5 (highlighted in blue and purple nodes) are involved in complement and coagulation cascade. Interacting O-glycoproteins such as Eng, Ecm1, Cadm1, Fbln5, Sema4d, Plxnb1, Bglap, Col3a1, Slc38a10, Glg1, and Ptprc (highlighted in green and gray nodes) are involved in bone development and remodeling.
Figure 8
Figure 8
Sequence motif analysis of GalNAc-T3 substrate O-glycosites. A, predominant amino acids experimentally identified 663 GalNAc-T3-specific O-glycosites, (B) 471 O-glycosites at threonine residues and (C) 188 at serine residues. D, frequency of amino acids flanking O-glycosite at the −1 and one position; X-S/T and S/T-X,(E) Heatmap of the frequency of amino acid residues at ±7 position flanking O-glycosites, the x-axis represents the ±7 amino acid position flanking O-glycosites (P0 position); y-axis represents amino acids (F) Euler diagram showing O-glycosites predicted by ISOGlyP and experimentally identified O-glycosites at threonine and serine residues. G, the sequence motif of ISOGlyP predicted 3750 GalNAc-T3 sites (EV > 1). H, sequence motif of experimentally identified 190 O-glycosites at threonine predicted by ISOGlyP. I, sequence motif of experimentally identified 290 O-glycosites at threonine NOT predicted by ISOGlyP. EV, enhancement value.
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