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Structural basis of mammalian high-mannose N-glycan processing by human gut Bacteroides.

ABSTRACT: The human gut microbiota plays a central role not only in regulating the metabolism of nutrients but also promoting immune homeostasis, immune responses and protection against pathogen colonization. The genome of the Gram-negative symbiont Bacteroides thetaiotaomicron, a dominant member of the human intestinal microbiota, encodes polysaccharide utilization loci PULs, the apparatus required to orchestrate the degradation of a specific glycan. EndoBT-3987 is a key endo-?-N-acetylglucosaminidase (ENGase) that initiates the degradation/processing of mammalian high-mannose-type (HM-type) N-glycans in the intestine. Here, we provide structural snapshots of EndoBT-3987, including the unliganded form, the EndoBT-3987-Man9GlcNAc2Asn substrate complex, and two EndoBT-3987-Man9GlcNAc and EndoBT-3987-Man5GlcNAc product complexes. In combination with alanine scanning mutagenesis and activity measurements we unveil the molecular mechanism of HM-type recognition and specificity for EndoBT-3987 and an important group of the GH18 ENGases, including EndoH, an enzyme extensively used in biotechnology, and for which the mechanism of substrate recognition was largely unknown.

SUBMITTER: Trastoy B

PROVIDER: S-EPMC7021837 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Structural basis of mammalian high-mannose N-glycan processing by human gut Bacteroides.

Trastoy Beatriz B Du Jonathan J JJ Klontz Erik H EH Li Chao C Cifuente Javier O JO Wang Lai-Xi LX Sundberg Eric J EJ Guerin Marcelo E ME

Nature communications 20200214 1

The human gut microbiota plays a central role not only in regulating the metabolism of nutrients but also promoting immune homeostasis, immune responses and protection against pathogen colonization. The genome of the Gram-negative symbiont Bacteroides thetaiotaomicron, a dominant member of the human intestinal microbiota, encodes polysaccharide utilization loci PULs, the apparatus required to orchestrate the degradation of a specific glycan. EndoBT-3987 is a key endo-β-N-acetylglucosaminidase (E ...[more]

PMID: 32060313

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Project description:N-glycosylation plays a key role in the quality of many therapeutic glycoprotein biologics. The biosynthesis reactions of these oligosaccharides are a type of network in which a relatively small number of enzymes give rise to a large number of N-glycans as the reaction intermediates and terminal products. Multiple glycans appear on the glycoprotein molecules and give rise to a heterogeneous product. Controlling the glycan distribution is critical to the quality control of the product. Understanding N-glycan biosynthesis and the etiology of microheterogeneity would provide physiological insights, and facilitate cellular engineering to enhance glycoprotein quality. We developed a mathematical model of glycan biosynthesis in the Golgi and analyzed the various reaction variables on the resulting glycan distribution. The Golgi model was modeled as four compartments in series. The mechanism of protein transport across the Golgi is still controversial. From the viewpoint of their holding time distribution characteristics, the two main hypothesized mechanisms, vesicular transport and Golgi maturation models, resemble four continuous mixing-tanks (4CSTR) and four plug-flow reactors (4PFR) in series, respectively. The two hypotheses were modeled accordingly and compared. The intrinsic reaction kinetics were first evaluated using a batch (or single PFR) reactor. A sufficient holding time is needed to produce terminally-processed glycans. Altering enzyme concentrations has a complex effect on the final glycan distribution, as the changes often affect many reaction steps in the network. Comparison of the glycan profiles predicted by the 4CSTR and 4PFR models points to the 4PFR system as more likely to be the true mechanism. To assess whether glycan heterogeneity can be eliminated in the biosynthesis of biotherapeutics the 4PFR model was further used to assess whether a homogeneous glycan profile can be created through metabolic engineering. We demonstrate by the spatial localization of enzymes to specific compartments all terminally processed N-glycans can be synthesized as homogeneous products with a sufficient holding time in the Golgi compartments. The model developed may serve as a guide to future engineering of glycoproteins.

Dataset Information

Structural basis of mammalian high-mannose N-glycan processing by human gut Bacteroides.

Publications

Structural basis of mammalian high-mannose N-glycan processing by human gut Bacteroides.

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