Recombinant Arabidopsis thaliana Probable beta-1,3-galactosyltransferase 1 (B3GALT1)
Description
Functional Role in N-Glycan Biosynthesis
B3GALT1 is indispensable for Le<sup>a</sup> formation, a key modification in plant glycoproteins. Its role includes:
Key Steps in Le<sup>a</sup> Biosynthesis
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Galactosylation: B3GALT1 adds β1,3-galactose to N-glycans (e.g., converting GnGn to Galβ1-3GnGn) .
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Fucosylation: α1,4-Fucosyltransferase (e.g., FUT13) attaches fucose to the galactosylated intermediate, forming Le<sup>a</sup> .
Experimental Evidence:
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Knockout Studies: Arabidopsis plants lacking GALT1 mRNA show no detectable Le<sup>a</sup> epitopes .
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Overexpression: Transgenic plants overexpressing GALT1 exhibit elevated Le<sup>a</sup> levels, confirmed via MALDI-TOF MS .
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In Vitro Activity: Recombinant GALT1 produces monogalactosylated (m/z = 2223) and digalactosylated (m/z = 2385) N-glycans when incubated with UDP-galactose and glycopeptide substrates .
Comparative Analysis with Mammalian Orthologs
B3GALT1 differs from mammalian B3GALTs in structure and substrate specificity:
Mammalian B3GALT1 (ENSG00000172318, UniProt Q9Y5Z6) shares conserved catalytic residues but lacks the lectin domain .
Research Applications and Significance
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Glycosylation Engineering: B3GALT1 enables precise modification of plant N-glycans, relevant for biotechnology (e.g., humanizing glycoproteins) .
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Evolutionary Insights: Phylogenetic studies reveal B3GALT1 as part of a plant-specific clade distinct from animal β1,3-galactosyltransferases .
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Diagnostic Tools: Anti-Le<sup>a</sup> antibodies (e.g., JIM84) and MALDI-TOF MS are used to validate B3GALT1 activity in transgenic plants .
Product Specs
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Target Background
Function: Recombinant Arabidopsis thaliana Probable beta-1,3-galactosyltransferase 1 (B3GALT1) is a beta-1,3-galactosyltransferase that catalyzes the transfer of galactose from UDP-galactose to substrates possessing a terminal glycosyl residue.
KEGG: ath:AT1G11730
UniGene: At.51577
Q&A
Basic Research Questions
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What is Arabidopsis thaliana B3GALT1 and what is its function?
B3GALT1 (GALACTOSYLTRANSFERASE1, At1g26810) is a unique β1,3-galactosyltransferase in Arabidopsis thaliana that plays a critical role in plant glycosylation pathways. This enzyme specifically catalyzes the transfer of β1,3-linked galactose residues to N-glycans, which is essential for the biosynthesis of Lewis a epitopes [Fucα1-4(Galβ1-3)GlcNAc-R] in plants . B3GALT1 is both necessary and sufficient for this galactosylation process, as plants lacking functional B3GALT1 show no detectable Lewis a structures on their glycoproteins . The enzyme belongs to the CAZy glycosyltransferase family GT31 and contains a characteristic galactosyltransferase domain (pfam 01762) along with a galactoside binding lectin domain (pfam 00337) .
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Where is B3GALT1 localized in plant cells?
Cell biological characterization using confocal laser scanning microscopy of transiently expressed B3GALT1-fluorescent protein fusions has revealed that B3GALT1 is exclusively localized within the Golgi apparatus of plant cells . This specific subcellular localization aligns perfectly with its physiological function in the glycosylation of proteins, as the Golgi apparatus serves as the primary site for complex glycan modifications in the secretory pathway . The precise positioning within the Golgi apparatus supports B3GALT1's role in the sequential processing of N-glycans during their transit through the secretory pathway before glycoproteins reach their final destinations .
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What are the structural domains of B3GALT1 and how do they contribute to function?
B3GALT1 possesses a multi-domain architecture that enables its specific enzymatic function:
| Domain | Function | Note |
|---|---|---|
| Galactosyltransferase domain (pfam 01762) | Catalyzes transfer of galactose from UDP-galactose to acceptor | Conserved across GT31 family members |
| Galactoside binding lectin domain (pfam 00337) | Likely involved in substrate recognition | Unique to plant B3GALTs; not found in mammalian homologs |
| N-terminal transmembrane domain | Anchors protein to Golgi membrane | Single-pass membrane protein |
| N-glycosylation sites | Protein stability and folding | Contains seven potential sites |
The protein contains several highly conserved residues within the galactosyltransferase domain that are putatively involved in substrate binding and catalysis . The unique combination of catalytic and lectin domains distinguishes B3GALT1 from other glycosyltransferases and may explain its specific role in Lewis a epitope formation.
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What is the expression pattern of B3GALT1 in different Arabidopsis tissues?
B3GALT1 exhibits distinct tissue-specific expression patterns that correlate with the presence of Lewis a epitopes:
| Tissue | B3GALT1 Expression | Lewis a Epitope Presence |
|---|---|---|
| Stems | High | Strong signal |
| Pedicels | Not specified | Strong signal |
| Nodes | Not specified | Strong signal |
| Siliques | Moderate | Low signal |
| Shoot apex | Not specified | Low signal |
| Flowers | Not specified | Low signal |
| Roots | Not specified | Low signal |
| Leaves | Low | Undetectable |
RT-PCR analyses reveal that B3GALT1 mRNA is detectable in various organs but displays higher transcript levels in stems compared to leaves . This expression pattern correlates with immunoblot analyses using JIM84 (a Lewis a-specific antibody), which show Lewis a epitopes predominantly in stems, pedicels, and nodes . This tissue-specific expression suggests B3GALT1 may have specialized roles in certain plant organs, possibly related to specific developmental or physiological processes.
