id: Q8NBJ5 gene_symbol: COLGALT1 product_type: PROTEIN status: COMPLETE taxon: id: NCBITaxon:9606 label: Homo sapiens description: COLGALT1 (collagen beta(1-O)galactosyltransferase 1; also known as GLT25D1) is a soluble enzyme of the endoplasmic reticulum lumen that catalyzes the first committed step of collagen O-linked glycosylation. It transfers galactose from UDP-alpha-D-galactose onto (5R)-5-hydroxy-L-lysine (hydroxylysine) residues of collagen, forming the beta(1-O)-linked Gal-O-hydroxylysine (EC 2.4.1.50). This galactose is then the acceptor for subsequent alpha1,2-glucosylation, generating the Glc(alpha1-2)Gal disaccharide that is strongly conserved across animal collagens. The enzyme belongs to glycosyltransferase family 25 (GT25), adopts a GT-A-like fold with metal-dependent (Mn2+) catalysis, and uses essential aspartate residues (D166/D168, D461/D463) for activity. COLGALT1 acts on multiple collagen types (including types I and IV) as well as the collagenous domain of mannose-binding lectin, and co-localizes in the early secretory pathway with the upstream lysyl hydroxylase PLOD3/LH3. As the predominant of two paralogous collagen galactosyltransferases (the other being COLGALT2/GLT25D2), it is broadly expressed and its loss reduces collagen glycosylation, causing intracellular collagen accumulation. Biallelic loss-of-function variants in COLGALT1 cause an autosomal recessive cerebral small vessel disease (brain small vessel disease 3). references: - id: GO_REF:0000033 title: Annotation inferences using phylogenetic trees findings: [] - id: GO_REF:0000044 title: Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping, accompanied by conservative changes to GO terms applied by UniProt findings: [] - id: GO_REF:0000120 title: Combined Automated Annotation using Multiple IEA Methods findings: [] - id: PMID:19075007 title: Core glycosylation of collagen is initiated by two beta(1-O)galactosyltransferases. findings: - statement: GLT25D1/COLGALT1 and GLT25D2 are beta(1-O)galactosyltransferases that initiate core glycosylation of collagen by transferring galactose to hydroxylysine residues; activity is the basis for EC 2.4.1.50 assignment, with KM ~18.7 uM for UDP-galactose. reference_section_type: RESULTS - id: PMID:19946888 title: Defining the membrane proteome of NK cells. findings: - statement: COLGALT1 was identified in a large-scale mass-spectrometry membrane-proteome screen of NK-like YTS cells; ~60% of identified proteins were judged not to be plausible integral membrane proteins but rather transiently membrane-associated. reference_section_type: ABSTRACT - id: PMID:20470363 title: The human collagen beta(1-O)galactosyltransferase, GLT25D1, is a soluble endoplasmic reticulum localized protein. findings: - statement: GLT25D1/COLGALT1 is directed to the ER lumen as a soluble protein (cleaved N-terminal signal sequence) and retained there via a C-terminal RDEL ER-retrieval signal; membrane floatation shows it is not an integral membrane protein. reference_section_type: RESULTS - statement: GLT25D1 co-localizes with its substrate mannose-binding lectin (MBL) and with the upstream lysyl hydroxylase 3 (LH3/PLOD3) in the early secretory pathway. reference_section_type: RESULTS - id: PMID:22216269 title: Identification of domains and amino acids essential to the collagen galactosyltransferase activity of GLT25D1. findings: - statement: Mutagenesis identified aspartate residues D166/D168 and D461/D463 as essential for collagen galactosyltransferase activity, consistent with a GT-A metal-dependent catalytic fold; KM ~29.9 uM for UDP-galactose. reference_section_type: RESULTS - id: PMID:27402836 title: Collagen Accumulation in Osteosarcoma Cells lacking GLT25D1 Collagen Galactosyltransferase. findings: - statement: CRISPR/Cas9 inactivation of GLT25D1 in SaOS-2 osteosarcoma cells decreased collagen glycosylation by up to 60%, increased collagen type I expression, and caused intracellular (ER) accumulation of collagen type I, with compensatory induction of GLT25D2; loss did not alter collagen folding or thermal stability. reference_section_type: RESULTS - statement: GLT25D1 is the main collagen galactosyltransferase isoform; GLT25D2 loss alone had no effect on collagen secretion, and clones lacking both could not be recovered, suggesting collagen glycosylation is essential for osteosarcoma cell viability. reference_section_type: DISCUSSION - id: PMID:30412317 title: Biallelic COLGALT1 variants are associated with cerebral small vessel disease. findings: - statement: Biallelic COLGALT1 variants (L151R, A154P, G377R) cause brain small vessel disease 3 (BSVD3); the L151R variant abolishes galactosyltransferase activity, and COLGALT1 is implicated in collagen type IV biosynthesis. reference_section_type: RESULTS - id: Reactome:R-HSA-1650814 title: Collagen biosynthesis and modifying enzymes findings: [] - id: Reactome:R-HSA-1981120 title: Galactosylation of collagen propeptide hydroxylysines by procollagen galactosyltransferases 1, 2 findings: [] - id: Reactome:R-HSA-8948228 title: COLGALT1,COLGALT2 bind Lysyl hydroxylated collagen propeptides findings: [] - id: Reactome:R-HSA-8948231 title: COLGALT1,COLGALT2:Galactosyl-hydroxylysyl collagen propeptides dissociates findings: [] existing_annotations: - term: id: GO:0050211 label: procollagen galactosyltransferase activity evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: enables review: summary: Phylogenetically inferred procollagen galactosyltransferase activity. This is the well-established core molecular function of COLGALT1, directly demonstrated experimentally for the human enzyme. action: ACCEPT reason: This is the core molecular function of COLGALT1, supported by direct biochemical characterization (EC 2.4.1.50) and mutagenesis. The IBA transfer is fully consistent with the experimental evidence. supported_by: - reference_id: PMID:19075007 supporting_text: >- Amino acid analysis of the products of GLT25D1 and GLT25D2 reactions confirmed the transfer of galactose to hydroxylysine residues. - term: id: GO:0005788 label: endoplasmic reticulum lumen evidence_type: IEA original_reference_id: GO_REF:0000044 qualifier: located_in review: summary: ER lumen localization mapped from UniProt subcellular location. COLGALT1 is a soluble ER-lumen protein, directly shown by immunofluorescence and membrane-floatation assays. action: ACCEPT reason: ER lumen is the correct site of action; this electronic mapping agrees with direct experimental localization evidence. supported_by: - reference_id: PMID:20470363 supporting_text: >- In agreement with the predictions our results show that GLT25D1 is directed to the ER lumen as a soluble protein and retained there. - term: id: GO:0050211 label: procollagen galactosyltransferase activity evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: enables review: summary: Electronically inferred procollagen galactosyltransferase activity (mapped from RHEA:12637 / EC 2.4.1.50). This is the experimentally validated core function. action: ACCEPT reason: Consistent with the experimentally demonstrated catalytic activity and EC assignment. supported_by: - reference_id: file:human/COLGALT1/COLGALT1-uniprot.txt supporting_text: EC=2.4.1.50; Reaction=(5R)-5-hydroxy-L-lysyl-[collagen] + UDP-alpha-D-galactose = (5R)-5-O-(beta-D-galactosyl)-5-hydroxy-L-lysyl-[collagen] + UDP + H(+); Xref=Rhea:RHEA:12637. - term: id: GO:0030199 label: collagen fibril organization evidence_type: TAS original_reference_id: Reactome:R-HSA-1650814 qualifier: involved_in review: summary: COLGALT1 contributes to collagen biosynthesis/modification, which is upstream of collagen fibril organization in the extracellular matrix. Its direct action is intracellular hydroxylysine galactosylation, not fibril assembly itself. action: KEEP_AS_NON_CORE reason: This is a downstream, indirect biological consequence of collagen glycosylation rather than the core molecular function. Glycosylation has been suggested to influence collagen crosslink formation and matrix organization, but the enzyme does not directly organize fibrils. Retaining as non-core captures the pathway context without overstating the role. supported_by: - reference_id: PMID:27402836 supporting_text: glycosylation may be involved in the organization of collagens in the extracellular space. Glycosylation has been suggested to regulate cross-link formation in fibrillar collagens. - term: id: GO:0050211 label: procollagen galactosyltransferase activity evidence_type: TAS original_reference_id: Reactome:R-HSA-1981120 qualifier: enables review: summary: Reactome-asserted procollagen galactosyltransferase activity, the core molecular function of COLGALT1. action: ACCEPT reason: Core molecular function, well supported by direct biochemical and mutagenesis evidence. supported_by: - reference_id: PMID:19075007 supporting_text: >- Amino acid analysis of the products of GLT25D1 and GLT25D2 reactions confirmed the transfer of galactose to hydroxylysine residues. - term: id: GO:0180062 label: protein O-linked glycosylation via galactose evidence_type: IMP original_reference_id: PMID:27402836 qualifier: involved_in review: summary: COLGALT1 performs the galactose-transfer step of collagen O-linked glycosylation onto hydroxylysine. Its loss reduces collagen glycosylation by up to 60% in osteosarcoma cells, directly implicating it in this process. action: ACCEPT reason: This accurately describes the biological process the enzyme directly carries out (O-linked galactosylation of hydroxylysine), supported by loss-of-function data. supported_by: - reference_id: PMID:27402836 supporting_text: Loss of GLT25D1 decreased collagen glycosylation by up to 60%. - term: id: GO:0050211 label: procollagen galactosyltransferase activity evidence_type: IMP original_reference_id: PMID:27402836 qualifier: enables review: summary: Loss of GLT25D1/COLGALT1 reduces collagen galactosylation, providing functional (mutant phenotype) support for its procollagen galactosyltransferase activity. action: ACCEPT reason: Core molecular function with direct loss-of-function support, corroborating the biochemical characterization. supported_by: - reference_id: PMID:27402836 supporting_text: Loss of GLT25D1 decreased collagen glycosylation by up to 60%. - term: id: GO:1904028 label: positive regulation of collagen fibril organization evidence_type: IMP original_reference_id: PMID:27402836 qualifier: involved_in review: summary: This annotation derives from loss-of-function studies in osteosarcoma cells, but those experiments measured collagen glycosylation, intracellular collagen accumulation, and collagen type I expression, not fibril organization. The same study reported that loss of GLT25D1 did not alter collagen folding or thermal stability. action: MARK_AS_OVER_ANNOTATED reason: The cited paper does not demonstrate positive regulation of collagen fibril organization; it showed intracellular collagen accumulation and unchanged folding/thermal stability. Any effect on fibril organization is indirect and speculative. This regulatory term over-interprets the available evidence. supported_by: - reference_id: PMID:27402836 supporting_text: Loss of GLT25D1 decreased collagen glycosylation by up to 60% but did not alter collagen folding and thermal stability. - term: id: GO:0016020 label: membrane evidence_type: HDA original_reference_id: PMID:19946888 qualifier: located_in review: summary: This high-throughput annotation comes from a mass-spectrometry membrane-proteome screen of NK-like YTS cells. COLGALT1 is a soluble ER-lumen protein (no transmembrane domain), and the screen itself noted that a large fraction of identified proteins were not plausible integral membrane proteins but transiently associated. action: MARK_AS_OVER_ANNOTATED reason: COLGALT1 is a soluble protein of the ER lumen, directly demonstrated by membrane-floatation and localization assays; it has a cleaved signal sequence and no transmembrane region. The membrane assignment reflects co-isolation in a proteomic screen rather than genuine membrane residence, and is contradicted by direct evidence. supported_by: - reference_id: PMID:20470363 supporting_text: The bulk of GLT25D1 is observed in the bottom fractions of the gradient, indicating the protein is not an integral membrane protein. - term: id: GO:0005788 label: endoplasmic reticulum lumen evidence_type: TAS original_reference_id: Reactome:R-HSA-1981120 qualifier: located_in review: summary: ER lumen localization asserted by Reactome, consistent with direct experimental evidence that COLGALT1 is a soluble ER-lumen protein. action: ACCEPT reason: Correct site of action, corroborated by direct localization data. supported_by: - reference_id: PMID:20470363 supporting_text: GLT25D1 is directed to the ER lumen as a soluble protein and retained there. - term: id: GO:0005788 label: endoplasmic reticulum lumen evidence_type: TAS original_reference_id: Reactome:R-HSA-8948228 qualifier: located_in review: summary: ER lumen localization asserted by Reactome (COLGALT1/COLGALT2 binding lysyl-hydroxylated collagen propeptides), consistent with the soluble ER-lumen localization of the enzyme. action: ACCEPT reason: Correct localization, corroborated by direct experimental evidence. supported_by: - reference_id: PMID:20470363 supporting_text: GLT25D1 is directed to the ER lumen as a soluble protein and retained there. - term: id: GO:0005788 label: endoplasmic reticulum lumen evidence_type: TAS original_reference_id: Reactome:R-HSA-8948231 qualifier: located_in review: summary: ER lumen localization asserted by Reactome, consistent with the experimentally established soluble ER-lumen residence of COLGALT1. action: ACCEPT reason: Correct localization, corroborated by direct experimental evidence. supported_by: - reference_id: PMID:20470363 supporting_text: GLT25D1 is directed to the ER lumen as a soluble protein and retained there. - term: id: GO:0005788 label: endoplasmic reticulum lumen evidence_type: IDA original_reference_id: PMID:20470363 qualifier: located_in review: summary: Direct immunofluorescence and membrane-floatation evidence that COLGALT1 is a soluble protein of the ER lumen, retained via a C-terminal RDEL signal, co-localizing with PLOD3/LH3 and MBL in the early secretory pathway. action: ACCEPT reason: This is the strongest, most direct evidence for the cellular location of COLGALT1 and represents its core site of action. supported_by: - reference_id: PMID:20470363 supporting_text: GLT25D1 is directed to the ER lumen as a soluble protein and retained there. - term: id: GO:0030145 label: manganese ion binding evidence_type: IC original_reference_id: PMID:22216269 qualifier: enables review: summary: Proposed annotation not present in the current GOA for COLGALT1. action: NEW reason: COLGALT1 belongs to glycosyltransferase family 25 with a GT-A-like fold and essential catalytic aspartate residues (D166/D168, D461/D463 identified by mutagenesis), characteristic of divalent-metal (Mn2+)-dependent glycosyltransferases. Mn2+ dependence is expected on family and structural grounds, although no direct experimental metal-binding annotation currently exists in GOA. supported_by: - reference_id: PMID:22216269 supporting_text: Mutagenesis identified aspartate residues D166/D168 and D461/D463 as essential for collagen galactosyltransferase activity, consistent with a GT-A metal-dependent catalytic fold. full_text_unavailable: true core_functions: - description: Collagen beta(1-O)galactosyltransferase that transfers galactose from UDP-galactose onto hydroxylysine residues of collagen in the ER lumen, catalyzing the first step of collagen O-linked glycosylation. supported_by: - reference_id: PMID:19075007 supporting_text: >- Amino acid analysis of the products of GLT25D1 and GLT25D2 reactions confirmed the transfer of galactose to hydroxylysine residues. - reference_id: PMID:27402836 supporting_text: Loss of GLT25D1 decreased collagen glycosylation by up to 60%. molecular_function: id: GO:0050211 label: procollagen galactosyltransferase activity directly_involved_in: - id: GO:0180062 label: protein O-linked glycosylation via galactose locations: - id: GO:0005788 label: endoplasmic reticulum lumen proposed_new_terms: [] suggested_questions: - question: Does COLGALT1 require Mn2+ (or another divalent cation) for catalysis, and which residues coordinate the metal in the recently solved GT25 structures? - question: To what extent are the collagen folding/secretion phenotypes of COLGALT1 loss tissue- and collagen-type-specific (e.g., type IV in cerebral vasculature vs. type I in bone), given paralogous compensation by COLGALT2? suggested_experiments: - description: Use the available cryo-EM and X-ray structures (e.g., PDB 8ZGE, 9EVJ) together with metal-substitution and site-directed mutagenesis to confirm the catalytic metal requirement and map the divalent-cation coordination site. - description: Generate tissue-specific or vascular COLGALT1 knockouts/knock-ins of BSVD3 variants and quantify collagen type IV glycosylation, basement membrane integrity, and small-vessel pathology to causally link enzymatic loss to disease.