Alvarez, Bernadette2025-08-292025-08-292025https://hdl.handle.net/1828/22694The human gut microbiota (HGM) plays a significant role in maintaining our overall health through its dynamic composition, ability to degrade recalcitrant nutrient sources, and production of metabolites that enable host-gut microbiota crosstalk. However, many aspects of HGM function remain poorly understood. Despite the highly diverse nature of the human diet, our understanding on how the HGM metabolises the glycan and peptide components of glycoproteins is limited. Here, we investigate how polysaccharide utilization locus 25 from Bacteroides caccae (BcPUL25) could target the glycosaminoglycan (GAG) and peptide components of aggrecan, a major dietary proteoglycan in animal cartilage. Through structure-function analyses of its encoded carbohydrate-active enzymes (CAZymes), we show that BcPUL25 targets desulfated chondroitin sulfate, or chondroitin, through a unique pathway that uses a carbohydrate dehydratase to prime saturated reducing end β-glucuronic acid residues for cleavage, allowing for more efficient GAG degradation by this PUL. Similarly, biochemical assessment of both BcM60_F and BcM60_G using mucin-specific FRET substrates revealed that BcM60_G can accommodate numerous glycan structures, whereas BcM60_F exhibited more stringent peptide specificity than its counterpart and could only bind to linear glycan moieties. All tested mucin O-glycan structures have been observed to decorate the aggrecan peptide backbone. Collectively, these findings establish B. caccae as a potential glycoprotein specialist and provides the molecular framework for exploring glycoprotein metabolism by the HGM. Overall, this research contributes toward our understanding of the molecular interplay within the trifecta of microbial composition, nutrient availability, and metabolite production. In the future, studies such as this will support, and eventually result in, the development of therapeutics that will alleviate HGM-influenced disease states.enAvailable to the World Wide WebHuman gut microbiota (HGM)Carbohydrate active enzymes (CAZymes)Proteoglycan degradationBacteroides caccaeM60-like peptidasesThesisAnalysis of chondroitin degradation by components of a Bacteroides caccae polysaccharide utilization locus. JBC. 2025 June 7; 10.1016/j.jbc.2025.110354