Identificación, clonación y análisis funcional de la fucosidasa GH95Fuc25 aislada del metagenoma de la microbiota intestinal de niños lactantes.

Abstract

[EN] Gut microbiota is a dynamic community that can be altered by the host diet as well as internal host factors. Breastfeeding modulates the composition of the gut microbiota. Human milk contains specific oligosaccharides (HMOs) that are not digested by infants and when they reach the intestine, they are metabolized by certain bacterial species, favoring their colonization and growth. HMOs consist of a combination of 5 monosaccharides: D-glucose, D-galactose, N-acetylglucosamine (GlcNAc), sialic acid and L-fucose. Residues of L-fucose are present at the non-reducing end of HMOs and other relevant glycans such as histo-blood group antigens (AGSs). Glycoconjugates of these AGSs present on the cell surfaces of the gut epithelium act as receptors for enteropathogens and as metabolic substrates for bacterias. The use of fucosylated oligosaccharides by these bacterias requires L-fucosidase enzymes. In this work sequence analysis of L-fucosidases from the GH95 family present in the metagenome of the infant gut microbiota, previously obtained in the laboratory, has been carried out. Based on sequence homology, a total of 27 L-fucosidases GH95 were identified and the gene that codes for the fucosidase named GH95Fuc25 has been cloned and expressed in Escherichia coli. The enzyme has been purified and its kinetic and biochemical properties have been evaluated using p-nitrophenyl L-fucopyranoside as substrate. Its specificity against natural fucosylated glycans has also been determined. GH95Fuc25 can efficiently hydrolyze blood group antigens type 1 and 2 and the HMOs 2'-fucosyllactose (2'FL) and 3-fucosyllactose (3FL). It also hydrolyzes fucose residues of human acid glycoprotein, bovine lactoferrin and porcine mucin. These results indicate that this enzyme shows substrate specificities towards HMOs, AGSs and natural glycoproteins, suggesting a great biotechnological potential.

[ES] La microbiota intestinal es una comunidad dinámica que puede ser alterada por la alimentación del hospedador así como por factores internos del mismo. La lactancia materna modula la composición de la microbiota intestinal. Así, la leche humana contiene oligosacáridos específicos (OLHs) que no son digeridos por los lactantes y llegan al intestino, donde son metabolizados por determinadas especies bacterianas, favoreciendo su colonización y crecimiento. Los OLHs consisten en una combinación de 5 monosacáridos: D-glucosa, D-galactosa, N-acetilglucosamina (GlcNAc), ácido siálico y L-fucosa. Los residuos de -L-fucosa están presentes en el extremo no reductor de los OLHs y de otros glicanos relevantes como los antígenos de los grupos histo-sanguíneos (AGSs). Glicoconjugados de estos AGSs presentes en las superficies celulares del epitelio intestinal actúan como receptores para enteropatógenos y también como sustratos metabólicos para las bacterias. La utilización de los oligosacáridos fucosilados por éstas requiere de enzimas L-fucosidasas. En el presente trabajo se ha realizado un análisis de las secuencias de las L-fucosidasas de la familia GH95 presentes en el metagenoma de la microbiota fecal de niños lactantes, obtenido anteriormente en el laboratorio. Basándose en homología de secuencia se identificaron un total de 27 L-fucosidasas GH95 y el gen que codifica para la L-fucosidasa denominada GH95Fuc25 se ha clonado y expresado en Escherichia coli. La enzima se ha purificado y se han evaluado sus propiedades cinéticas y bioquímicas utilizando como sustrato p-nitrofenil-L-fucopiranosido. También se ha determinado su especificidad frente a glicanos fucosilados naturales. GH95Fuc25 es capaz de hidrolizar de forma eficiente los antígenos del grupo sanguíneo H tipo 1 y tipo 2, y los OLHs 2'-fucosil-lactosa (2'FL) y 3-fucosil-lactosa (3FL). También hidroliza residuos de fucosa de las glicoproteínas ácida -1 humana, lactoferrina bovina y la mucina porcina. Los resultados obtenidos indican que esta enzima muestra especificidades de sustrato hacia OLHs, AGSs y glicoproteínas naturales, lo que sugiere un gran potencial biotecnológico. Este trabajo se relaciona con los siguientes ODS de la Agenda 2030: Salud y Bienestar (ODS 3); Producción y consumo responsables (ODS 12).

[EN] Gut microbiota is a dynamic community that can be altered by the host diet as well as internal host factors. Breastfeeding modulates the composition of the gut microbiota. Human milk contains specific oligosaccharides (HMOs) that are not digested by infants and when they reach the intestine, they are metabolized by certain bacterial species, favoring their colonization and growth. HMOs consist of a combination of 5 monosaccharides: D-glucose, D-galactose, N-acetylglucosamine (GlcNAc), sialic acid and L-fucose. Residues of ¿-L-fucose are present at the non-reducing end of HMOs and other relevant glycans such as histo-blood group antigens (AGSs). Glycoconjugates of these AGSs present on the cell surfaces of the gut epithelium act as receptors for enteropathogens and as metabolic substrates for bacterias. The use of fucosylated oligosaccharides by these bacterias requires ¿-L-fucosidase enzymes. In this work sequence analysis of ¿-L-fucosidases from the GH95 family present in the metagenome of the infant gut microbiota, previously obtained in the laboratory, has been carried out. Based on sequence homology, a total of 27 ¿-L-fucosidases GH95 were identified and the gene that codes for the fucosidase named GH95Fuc25 has been cloned and expressed in Escherichia coli. The enzyme has been purified and its kinetic and biochemical properties have been evaluated using p-nitrophenyl-¿-L-fucopyranoside as substrate. Its specificity against natural fucosylated glycans has also been determined. GH95Fuc25 can efficiently hydrolyze blood group antigens type 1 and 2 and the HMOs 2'-fucosyllactose (2'FL) and 3-fucosyllactose (3FL). It also hydrolyzes fucose residues of human ¿-acid glycoprotein, bovine lactoferrin and porcine mucin. These results indicate that this enzyme shows substrate specificities towards HMOs, AGSs and natural glycoproteins, suggesting a great biotechnological potential.