Dataset Information

IgA1 hinge-region clustered glycan fidelity is established early during semi-ordered glycosylation by GalNAc-T2.

ABSTRACT: GalNAc-type O-glycans are often added to proteins post-translationally in a clustered manner in repeat regions of proteins, such as mucins and IgA1. Observed IgA1 glycosylation patterns show that glycans occur at similar sites with similar structures. It is not clear how the sites and number of glycans added to IgA1, or other proteins, can follow a conservative process. GalNAc-transferases initiate GalNAc-type glycosylation. In IgA nephropathy, an autoimmune disease, the sites and O-glycan structures of IgA1 hinge-region are altered, giving rise to a glycan autoantigen. To better understand how GalNAc-transferases determine sites and densities of clustered O-glycans, we used IgA1 hinge-region (HR) segment as a probe. Using LC-MS, we demonstrated a semi-ordered process of glycosylation by GalNAc-T2 towards the IgA1 HR. The catalytic domain was responsible for selection of four initial sites based on amino-acid sequence recognition. Both catalytic and lectin domains were involved in multiple second site-selections, each dependent on initial site-selection. Our data demonstrated that multiple start-sites and follow-up pathways were key to increasing the number of glycans added. The lectin domain predominately enhanced IgA1 HR glycan density by increasing synthesis pathway exploration by GalNAc-T2. Our data indicated a link between site-specific glycan addition and clustered glycan density that defines a mechanism of how conserved clustered O-glycosylation patterns and glycoform populations of IgA1 can be controlled by GalNAc-T2. Together, these findings characterized a correlation between glycosylation pathway diversity and glycosylation density, revealing mechanisms by which a single GalNAc-T isozyme can limit and define glycan heterogeneity in a disease-relevant context.

SUBMITTER: Stewart TJ

PROVIDER: S-EPMC6583770 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Publications

IgA1 hinge-region clustered glycan fidelity is established early during semi-ordered glycosylation by GalNAc-T2.

Stewart Tyler J TJ Takahashi Kazuo K Whitaker Robert H RH Raska Milan M Placzek William J WJ Novak Jan J Renfrow Matthew B MB

Glycobiology 20190701 7

GalNAc-type O-glycans are often added to proteins post-translationally in a clustered manner in repeat regions of proteins, such as mucins and IgA1. Observed IgA1 glycosylation patterns show that glycans occur at similar sites with similar structures. It is not clear how the sites and number of glycans added to IgA1, or other proteins, can follow a conservative process. GalNAc-transferases initiate GalNAc-type glycosylation. In IgA nephropathy, an autoimmune disease, the sites and O-glycan struc ...[more]

PMID: 30759204

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Project description:The polypeptide N-acetylgalactosaminyl transferase (GalNAc-T) enzyme family initiates O-linked mucin-type glycosylation. The family constitutes 20 isoenzymes in humans. 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 (19x19) peptides with previously characterized experimental GalNAc-T2 glycosylation efficiencies. The calculations recapitulated experimental specificity data, successfully discriminating between glycosylatable and non-glycosylatable peptides with a probability of 96.5% (ROC-AUC score), a balanced accuracy of 85.5% and a false positive rate of 7.3%. 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.

Dataset Information

IgA1 hinge-region clustered glycan fidelity is established early during semi-ordered glycosylation by GalNAc-T2.

Publications

IgA1 hinge-region clustered glycan fidelity is established early during semi-ordered glycosylation by GalNAc-T2.

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