Dataset Information

It All Starts with a Sandwich: Identification of Sialidases with Trans-Glycosylation Activity.

ABSTRACT: Sialidases (3.2.1.18) may exhibit trans-sialidase activity to catalyze sialylation of lactose if the active site topology is congruent with that of the Trypanosoma cruzi trans-sialidase (EC 2.4.1.-). The present work was undertaken to test the hypothesis that a particular aromatic sandwich structure of two amino acids proximal to the active site of the T. cruzi trans-sialidase infers trans-sialidase activity. On this basis, four enzymes with putative trans-sialidase activity were identified through an iterative alignment from 2909 native sialidases available in GenBank, which were cloned and expressed in Escherichia coli. Of these, one enzyme, SialH, derived from Haemophilus parasuis had an aromatic sandwich structure on the protein surface facing the end of the catalytic site (Phe168; Trp366), and was indeed found to exhibit trans-sialidase activity. SialH catalyzed production of the human milk oligosaccharide 3'-sialyllactose as well as the novel trans-sialylation product 3-sialyllactose using casein glycomacropeptide as sialyl donor and lactose as acceptor. The findings corroborated that Tyr119 and Trp312 in the T. cruzi trans-sialidase are part of an aromatic sandwich structure that confers trans-sialylation activity for lactose sialylation. The in silico identification of trans-glycosidase activity by rational active site topology alignment thus proved to be a quick tool for selecting putative trans-sialidases amongst a large group of glycosyl hydrolases. The approach moreover provided data that help understand structure-function relations of trans-sialidases.

SUBMITTER: Nordvang RT

PROVIDER: S-EPMC4930215 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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It All Starts with a Sandwich: Identification of Sialidases with Trans-Glycosylation Activity.

Nordvang Rune T RT Nyffenegger Christian C Holck Jesper J Jers Carsten C Zeuner Birgitte B Sundekilde Ulrik K UK Meyer Anne S AS Mikkelsen Jørn D JD

PloS one 20160701 7

Sialidases (3.2.1.18) may exhibit trans-sialidase activity to catalyze sialylation of lactose if the active site topology is congruent with that of the Trypanosoma cruzi trans-sialidase (EC 2.4.1.-). The present work was undertaken to test the hypothesis that a particular aromatic sandwich structure of two amino acids proximal to the active site of the T. cruzi trans-sialidase infers trans-sialidase activity. On this basis, four enzymes with putative trans-sialidase activity were identified thro ...[more]

PMID: 27367145

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Project description:?-Glutamyl transpeptidase 1 (GGT1) is an N-glycosylated membrane protein that catabolizes extracellular glutathione and other ?-glutamyl-containing substrates. In a variety of disease states, including tumor formation, the enzyme is shed from the surface of the cell and can be detected in serum. The structures of the N-glycans on human GGT1 (hGGT1) have been shown to be tissue-specific. Tumor-specific changes in the glycans have also been observed, suggesting that the N-glycans on hGGT1 would be an important biomarker for detecting tumors and monitoring their progression during treatment. However, the large quantities of purified protein required to fully characterize the carbohydrate content poses a significant challenge for biomarker development. Herein, we investigated a new antibody-lectin sandwich array (ALSA) platform to determine whether this microanalytical technique could be applied to the characterization of N-glycan content of hGGT1 in complex biological samples.Our data show that hGGT1 can be isolated from detergent extracted membrane proteins by binding to the ALSA platform. Probing hGGT1 with lectins enables characterization of the N-glycans. We probed hGGT1 from normal human liver tissue, normal human kidney tissue, and hGGT1 expressed in the yeast Pichia pastoris. The lectin binding patterns obtained with the ALSA platform are consistent with the hGGT1 N-glycan composition obtained from previous large-scale hGGT1 N-glycan characterizations from these sources. We also validate the implementation of the Microcystis aeruginosa lectin, microvirin, in this platform and provide refined evidence for its efficacy in specifically recognizing high-mannose-type N-glycans, a class of carbohydrate modification that is distinctive of hGGT1 expressed by many tumors.Using this microanalytical approach, we provide proof-of-concept for the implementation of ALSA in conducting high-throughput studies aimed at investigating disease-related changes in the glycosylation patterns on hGGT1 with the goal of enhancing clinical diagnoses and targeted treatment regimens.

Dataset Information

It All Starts with a Sandwich: Identification of Sialidases with Trans-Glycosylation Activity.

Publications

It All Starts with a Sandwich: Identification of Sialidases with Trans-Glycosylation Activity.

Similar Datasets

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