title: A diverse range of bacterial and eukaryotic chitinases hydrolyzes the LacNAc (Galβ1-4GlcNAc) and LacdiNAc (GalNAcβ1-4GlcNAc) motifs found on vertebrate and insect cells.
abstract: There is emerging evidence that chitinases have additional functions beyond degrading environmental chitin, such as involvement in innate and acquired immune responses, tissue remodeling, fibrosis, and serving as virulence factors of bacterial pathogens. We have recently shown that both the human chitotriosidase and a chitinase from Salmonella enterica serovar Typhimurium hydrolyze LacNAc from Galβ1-4GlcNAcβ-tetramethylrhodamine (LacNAc-TMR (Galβ1-4GlcNAcβ(CH2)8CONH(CH2)2NHCO-TMR)), a fluorescently labeled model substrate for glycans found in mammals. In this study we have examined the binding affinities of the Salmonella chitinase by carbohydrate microarray screening and found that it binds to a range of compounds, including five that contain LacNAc structures. We have further examined the hydrolytic specificity of this enzyme and chitinases from Sodalis glossinidius and Polysphondylium pallidum, which are phylogenetically related to the Salmonella chitinase, as well as unrelated chitinases from Listeria monocytogenes using the fluorescently labeled substrate analogs LacdiNAc-TMR (GalNAcβ1-4GlcNAcβ-TMR), LacNAc-TMR, and LacNAcβ1-6LacNAcβ-TMR. We found that all chitinases examined hydrolyzed LacdiNAc from the TMR aglycone to various degrees, whereas they were less active toward LacNAc-TMR conjugates. LacdiNAc is found in the mammalian glycome and is a common motif in invertebrate glycans. This substrate specificity was evident for chitinases of different phylogenetic origins. Three of the chitinases also hydrolyzed the β1-6 bond in LacNAcβ1-6LacNAcβ-TMR, an activity that is of potential importance in relation to mammalian glycans. The enzymatic affinities for these mammalian-like structures suggest additional functional roles of chitinases beyond chitin hydrolysis.
authors.affiliation: From the Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Grønnegaardsvej 10, 1870 Frederiksberg C., Denmark.
authors.forename: Rikki F
authors.initials: RF
authors.lastname: Frederiksen
authors.affiliation: Carlsberg Laboratory, Gamle Carlsberg Vej 10, 1799 Copenhagen V, Denmark.
authors.forename: Yayoi
authors.initials: Y
authors.lastname: Yoshimura
authors.affiliation: From the Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Grønnegaardsvej 10, 1870 Frederiksberg C., Denmark, Carlsberg Laboratory, Gamle Carlsberg Vej 10, 1799 Copenhagen V, Denmark.
authors.forename: Birgit G
authors.initials: BG
authors.lastname: Storgaard
authors.affiliation: From the Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Grønnegaardsvej 10, 1870 Frederiksberg C., Denmark.
authors.forename: Dafni K
authors.initials: DK
authors.lastname: Paspaliari
authors.affiliation: Carlsberg Laboratory, Gamle Carlsberg Vej 10, 1799 Copenhagen V, Denmark.
authors.forename: Bent O
authors.initials: BO
authors.lastname: Petersen
authors.affiliation: Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Kbh. N., Denmark.
authors.forename: Kowa
authors.initials: K
authors.lastname: Chen
authors.affiliation: From the Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Grønnegaardsvej 10, 1870 Frederiksberg C., Denmark.
authors.forename: Tanja
authors.initials: T
authors.lastname: Larsen
authors.affiliation: Carlsberg Laboratory, Gamle Carlsberg Vej 10, 1799 Copenhagen V, Denmark.
authors.forename: Jens Ø
authors.initials: JØ
authors.lastname: Duus
authors.affiliation: From the Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Grønnegaardsvej 10, 1870 Frederiksberg C., Denmark.
authors.forename: Hanne
authors.initials: H
authors.lastname: Ingmer
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moskow 117997, Russian Federation.
authors.forename: Nicolai V
authors.initials: NV
authors.lastname: Bovin
authors.affiliation: Gesellschaft zur Förderung der Analytischen Wissenschaften e.V., ISAS-Leibnitz Institute for Analytical Sciences, Otto-Hahn-Strasse 6b, D-44227 Dortmund, Germany, and.
authors.forename: Ulrika
authors.initials: U
authors.lastname: Westerlind
authors.affiliation: Department of Chemistry, University of Copenhagen, 6:4:T422, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.
authors.forename: Ola
authors.initials: O
authors.lastname: Blixt
authors.affiliation: Carlsberg Laboratory, Gamle Carlsberg Vej 10, 1799 Copenhagen V, Denmark.
authors.forename: Monica M
authors.initials: MM
authors.lastname: Palcic
authors.affiliation: From the Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Grønnegaardsvej 10, 1870 Frederiksberg C., Denmark, jjl@sund.ku.dk.
authors.forename: Jørgen J
authors.initials: JJ
authors.lastname: Leisner
mesh_terms: D000606:Amino Sugars / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D002240:Carbohydrate Sequence; D002686:Chitin / Q000737:chemistry / Q000378:metabolism; D002688:Chitinases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D014644:Genetic Variation; D006801:Humans; D006868:Hydrolysis; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism*; D007313:Insecta; D007700:Kinetics; D007785:Lactose / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010802:Phylogeny; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D012235:Rhodamines / Q000737:chemistry / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D014714:Vertebrates
chemical_list: D000606:Amino Sugars; D001426:Bacterial Proteins; D019476:Insect Proteins; D011134:Polysaccharides; D012235:Rhodamines; C093701:N-acetylgalactosaminyl-1-4-N-acetylglucosamine; D002686:Chitin; C000458:N-acetyllactosamine; C005358:tetramethylrhodamine; D002688:Chitinases; D007785:Lactose
keywords: Adhesion; Chitinase; Family 18 Glycosidases; GlcNAc-containing Substrates; Glycoconjugate; Kinetics; Microarray