Molecular basis of the degradation and metabolism of N-glycans by the probiotic bacterium Bifidobacterium longum

Author(s)

Rosa Lorizolla Cordeiro

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2020-11-30

Examining board members

Mário Tyago Murakami; Celso Eduardo Benedetti; Glaucius Oliva; Shaker Chuck Farah; Beatriz Gomes Guimarães

• Advisor: Mário Tyago Murakami

Abstract

Bifidobacteria colonize the human gut and confer several advantages to our health, including the modulation of immune system and protection against pathogens. To survive in a highly resource-competitive environment, bifidobacteria have developed strategies to uptake and break down non-edible carbohydrates by the host. Some of these carbohydrates are produced by the host itself, such as the N-glycans present in glycoproteins found in the intestinal lumen. Bifidobacterium longum subsp. longum NCC2705 uses N-glycans as a carbon source; however, the molecular basis for the recognition and deconstruction of these carbohydrates were until then partially understood. In this work, we elucidate most of the molecular mechanisms of N-glycan degradation by B. longum deciphering the biochemical role of 5 glycosidic hydrolases that act in cascade for the release of simple sugars from these complex carbohydrates. In some cases, it was possible to unveil their three-dimensional structures by X-ray crystallography or cryo-electron microscopy (Cryo-EM), which revealed new structural elements for the recognition of N-glycans. A beta-mannosidase from the GH5 family of glycosyl hydrolases had an unknown function and showed to be highly specialized for the recognition of the disaccharide Man-beta-1,4-GlcNAc, which composes the universal core of eukaryotic N-glycans. Its specificity is dictated by a molecular architecture found only in this enzyme subfamily, which comprises a swapped tryptophan residue, a C-terminal region with conformation induced by the substrate and a Rossmann subdomain. Three alpha-mannosidases from the GH38 family are complementary in removing mannose residues from the antennas of N-glycans. Two divergent loops between these enzymes delimit their active sites and seem to determine their substrate preference. Furthermore, the presence of an alpha-glucosidase in the system indicates that B. longum is also capable of using immature N-glycosylations as nutrients, found on the intestinal epithelium. Finally, we discovered a metabolic route for mannose, the only monosaccharide whose metabolism was yet elusive in Bifidobacteria. We show that B. longum metabolizes mannose using a rare metabolic pathway involving a novel isomerase. Thus, in this thesis, we present the molecular basis of N-glycans depolymerization in B. longum and a putative metabolic route for mannose. These findings can be used to develop methods for the detection and treatment of diseases related to N-glycosylations, or even as molecular tools to characterize the structure of unknown N-glycans. Besides, this work provides biochemical and mechanistic information about the bacterium-host interaction of one of the most important bacterial genera for human health, which has co-evolved with humans over thousands of years (AU)

• FAPESP's process: 16/00740-2 - Mechanistic bases of evolutionary adaptation to temperature and specificity of glycoside hydrolases belonging to novel GH5 subfamilies
• Grantee: Rosa Lorizolla Cordeiro
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)