The structure of the colorectal cancer-associated enzyme GalNAc-T12 reveals how nonconserved residues dictate its function

Fernandez, Amy J.; Daniel, Earnest James Paul; Mahajan, Sai Pooja; Gray, Jeffrey J.; Gerken, Thomas A.; Tabak, Lawrence A.; Samara, N.L.

doi:10.1073/pnas.1902211116

Cited by 22 publications

(25 citation statements)

References 31 publications

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“…As the tactic relies on mutating gatekeeper residues that are conserved throughout the family, in principle, it should be applicable to other isoenzymes. Multiple GalNAc-Ts have been crystallized over the last years [ 43 , 130∗ , 131∗ , 132∗ , 133∗ ], and available structures seem to confirm this notion. The field of chemical glycoproteomics is rapidly evolving, constantly increasing the sensitivity of MS analysis.…”

Section: Introductionmentioning

confidence: 92%

Generating orthogonal glycosyltransferase and nucleotide sugar pairs as next-generation glycobiology tools

Cioce

Malaker

Schumann

2021

Current Opinion in Chemical Biology

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Protein glycosylation fundamentally impacts biological processes. Nontemplated biosynthesis introduces unparalleled complexity into glycans that needs tools to understand their roles in physiology. The era of quantitative biology is a great opportunity to unravel these roles, especially by mass spectrometry glycoproteomics. However, with high sensitivity come stringent requirements on tool specificity. Bioorthogonal metabolic labeling reagents have been fundamental to studying the cell surface glycoproteome but typically enter a range of different glycans and are thus of limited specificity. Here, we discuss the generation of metabolic ‘precision tools’ to study particular subtypes of the glycome. A chemical biology tactic termed bump-and-hole engineering generates mutant glycosyltransferases that specifically accommodate bioorthogonal monosaccharides as an enabling technique of glycobiology. We review the groundbreaking discoveries that have led to applying the tactic in the living cell and the implications in the context of current developments in mass spectrometry glycoproteomics.

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Section: Introductionmentioning

confidence: 92%

Generating orthogonal glycosyltransferase and nucleotide sugar pairs as next-generation glycobiology tools

Cioce

Malaker

Schumann

2021

Current Opinion in Chemical Biology

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“…Expression of recombinant PGANT9A and PGANT9B was performed either using P. pastoris or COS7 cells as described previously (21,38). Peptide substrates were synthesized by Peptide 2.0.…”

Section: Enzyme Assaysmentioning

confidence: 99%

“…Structurally, members of this family are similar, with each having a conserved catalytic domain responsible for binding donor (UDP-GalNAc) and acceptor (proteins or peptides) substrates to coordinate GalNAc transfer, as well as a separate ricin-like lectin domain that potentially recognizes an extant GalNAc on previously glycosylated substrates (17,18). The catalytic domain contains a catalytic flexible gating loop that becomes ordered and adopts a closed conformation upon acceptor and donor substrate binding (19)(20)(21)(22)(23). The lectin domain is unique to the GALNT enzyme family and consists of three repeats (a, b, and g) that can potentially bind to previously glycosylated substrates.…”

mentioning

confidence: 99%

“…The lectin domain is unique to the GALNT enzyme family and consists of three repeats (a, b, and g) that can potentially bind to previously glycosylated substrates. Such binding positions the acceptor site of a glycopeptide substrate into the catalytic domain to facilitate the glycosylation of additional, more distant sites (17)(18)(19)(21)(22)(23)(24)(25). Crystal structures of GALNT2, GALNT3, GALNT4, and GALNT12 bound to glycopeptide substrates show the glycopeptide-Thr-GalNAc bound to the a repeat of the lectin domain in an identical manner to help position the acceptor threonine into the active site of the catalytic domain at a distance (5-17 residues) from the lectin-bound glycopeptide-Thr-GalNAc (19)(20)(21)(22).…”

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confidence: 99%

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Differential splicing of the lectin domain of an O-glycosyltransferase modulates both peptide and glycopeptide preferences

May

Syed

et al. 2020

Journal of Biological Chemistry

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Mucin-type O-glycosylation is an essential post-translational modification required for protein secretion, extracellular matrix formation and organ growth. O-glycosylation is initiated by a large family of enzymes (GALNTs in mammals and PGANTs in Drosophila) that catalyze the addition of N-acetylgalactosamine (GalNAc) onto the hydroxyl groups of serines or threonines in protein substrates. These enzymes contain 2 functional domains; a catalytic domain and a C-terminal ricin-like lectin domain comprised of 3 potential GalNAc recognition repeats termed α, β and γ. The catalytic domain is responsible for binding donor and acceptor substrates and catalyzing transfer of GalNAc, while the lectin domain recognizes more distant extant GalNAc on previously glycosylated substrates. We previously demonstrated a novel role for the α repeat of lectin domain in influencing charged peptide preferences. Here, we further interrogate how the differentially spliced α repeat of the PGANT9A and PGANT9B O-glycosyltransferases confers distinct preferences for a variety of endogenous substrates. Through biochemical analyses and in silico modeling using preferred substrates, we find that a combination of charged residues within the α repeat and charged residues in the flexible gating loop of the catalytic domain distinctively influence the peptide substrate preferences of each splice variant. Moreover, PGANT9A and PGANT9B also display unique glycopeptide preferences. These data illustrate how changes within the non-catalytic lectin domain can alter the recognition of both peptide and glycopeptide substrates. Overall, our results elucidate a novel mechanism for modulating substrate preferences of O-glycosyltransferases via alternative splicing within specific subregions of functional domains.

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“…Computational studies can pinpoint key positions and structural motifs on an isoform that contribute to peptide substrate specificity. These sequence and structural motifs can be studied across isoforms to reveal more general patterns, to modulate enzyme specificity, and to gain insight into the consequences of enzyme and substrate mutations implicated in aberrant glycosylation, (e.g., colorectal cancer associated mutations of GalNAc-T12 17 ) paving the way for rational design of specific drugs/inhibitors. In this work, we seek to understand the sequence and structural motifs that determine the peptide substrate preferences for the GalNAc-T2 isoform.…”

Section: Introductionmentioning

confidence: 99%

Structural basis for peptide substrate specificities of glycosyltransferase GalNAc-T2

Mahajan

Srinivasan

Labonte

et al. 2020

Preprint

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The polypeptide N-acetylgalactosaminyl transferase (GalNAc-T) enzyme family initiates O-linked mucin-type glycosylation. The family constitutes 20 isozymes in humans—an unusually large number—unique to O-glycosylation. GalNAc-Ts exhibit both redundancy and finely tuned specificity for a wide range of peptide substrates. In this work, we deciphered the sequence and structural motifs that determine the peptide substrate preferences for the GalNAc-T2 isoform. Our approach involved sampling and characterization of peptide–enzyme conformations obtained from Rosetta Monte Carlo-minimization–based flexible docking. We computationally scanned 19 amino acid residues at positions –1 and +1 of an eight-residue peptide substrate, which comprised a dataset of 361 (19×19) peptides with previously characterized experimental GalNAc-T2 glycosylation efficiencies. The calculations recapitulated experimental specificity data, successfully discriminating between glycosylatable and non-glycosylatable peptides with an accuracy of 89% and a ROC-AUC score of 0.965. The glycosylatable peptide substrates viz. peptides with proline, serine, threonine, and alanine at the –1 position of the peptide preferentially exhibited cognate sequon-like conformations. The preference for specific residues at the −1 position of the peptide was regulated by enzyme residues R362, K363, Q364, H365 and W331, which modulate the pocket size and specific enzyme-peptide interactions. For the +1 position of the peptide, enzyme residues K281 and K363 formed gating interactions with aromatics and glutamines at the +1 position of the peptide, leading to modes of peptide-binding sub-optimal for catalysis. Overall, our work revealed enzyme features that lead to the finely tuned specificity observed for a broad range of peptide substrates for the GalNAc-T2 enzyme. We anticipate that the key sequence and structural motifs can be extended to analyze specificities of other isoforms of the GalNAc-T family and can be used to guide design of variants with tailored specificity.

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The structure of the colorectal cancer-associated enzyme GalNAc-T12 reveals how nonconserved residues dictate its function

Cited by 22 publications

References 31 publications

Generating orthogonal glycosyltransferase and nucleotide sugar pairs as next-generation glycobiology tools

Generating orthogonal glycosyltransferase and nucleotide sugar pairs as next-generation glycobiology tools

Differential splicing of the lectin domain of an O-glycosyltransferase modulates both peptide and glycopeptide preferences

Structural basis for peptide substrate specificities of glycosyltransferase GalNAc-T2

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