Cloning, functional expression and purification of endo-β-galactosidase from Flavobacterium keratolyticus

Gal␣1-3Gal is the major xenoantigenic epitope responsible for hyperacute rejection upon pig to human xenotransplantation. Endo-␤-galactosidase C from Clostridium perfringens destroys the antigenic epitope by cleaving the ␤-galactosidic linkage in the Gal␣1-3Gal␤1-4GlcNAc structure. Based on partial peptide sequences of the enzyme, we molecularly cloned the enzyme gene, which encodes a protein with a predicted molecular mass of about 93 kDa. The deduced protein sequence of the enzyme has limited homology in the C-terminal half with endo-␤-galactosidase from Flavobacterium keratolyticus and ␤-1,3-glucanases. The enzyme expressed in Escherichia coli removed the ␣-galactosyl epitope nearly completely from pig erythrocytes and from pig aortic endothelial cells. The enzyme-treated endothelial cells in culture were greatly reduced in cell surface antigens, which were recognized by IgM, IgG, or IgA in human sera, and became much less susceptible to complement-mediated cytotoxicity caused by human sera. When the pig kidney was perfused with the enzyme, the vascular endothelial cells became virtually devoid of the ␣-galactosyl epitope, with concomitant decrease in binding to IgM in human plasma. These results demonstrated that the recombinant endo-␤-galactosidase C is a valuable aid in xenotransplantation.

Section: Cloning and Structural Analysis Of Endo-␤-galactosidase C-sdsupporting

confidence: 77%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Molecular Cloning of Endo-β-galactosidase C and Its Application in Removing α-Galactosyl Xenoantigen from Blood Vessels in the Pig Kidney

Ogawa¹,

Muramatsu²,

Kobayashi³

et al. 2000

“…They are: (i) the endo-␤-galactosidase that cleaves the endo-␤-galactosyl linkages in polylactosaminoglycans (23)(24)(25)(26)(27); (ii) the endo-␤-galactosidase that releases blood group A and B trisaccharides from blood group A and B substances (28); and (iii) the endo-␤-galactosidase (Endo-Gal-C) that releases the Gal␣133Gal from the xenoantigen Gal␣133Gal␤13 R (29). The endo-␤-galactosidase presented in this report is distinct from the aforementioned three endo-␤-galactosidases.…”

Section: Discussionmentioning

confidence: 99%

A Novel Endo-β-galactosidase from Clostridium perfringens That Liberates the Disaccharide GlcNAcα1→4Gal from Glycans Specifically Expressed in the Gastric Gland Mucous Cell-type Mucin

Ashida¹,

Anderson²,

Nakayama³

et al. 2001

We found that commercially available sialidases prepared from Clostridium perfringens ATCC10543 were contaminated with an endoglycosidase capable of releasing the disaccharide GlcNAc␣134Gal from glycans expressed in the gastric gland mucous cell-type mucin. We have isolated this enzyme in electrophoretically homogeneous form from the culture supernatant of this organism by ammonium sulfate precipitation followed by affinity chromatography using a Sephacryl S-200 HR column. The enzyme was specifically retained by and eluted from the column with methyl-␣-Glc. By NMR spectroscopy, the structure of the disaccharide released from porcine gastric mucin by this enzyme was established to be GlcNAc␣134Gal. The specificity of this enzyme as an endo-␤-galactosidase was established by analyzing the liberation of GlcNAc␣134Gal from GlcNAc␣134Gal␤134GlcNAc␤136(GlcNAc␣13 4Gal␤133)GalNAc-ol by mass spectrometry. Because this novel endo-␤-galactosidase specifically releases the GlcNAc␣134Gal moiety from porcine gastric mucin, we propose to call this enzyme a GlcNAc␣134Gal-releasing endo-␤-galactosidase (Endo-␤-Gal GnGa ). Endo-␤-Gal GnGa was found to remove the GlcNAc␣134Gal epitope expressed in gastric adenocarcinoma AGS cells transfected with ␣1,4-N-acetylglucosaminyltransferase cDNA. Endo-␤-Gal GnGa should become useful for studying the structure and function of glycoconjugates containing the terminal GlcNAc␣134Gal epitope.

“…The CBM16 polypeptides constitute a large family of 59 members that are linked to diverse catalytic domains (17)(18)(19)(20)(21), suggesting that these CBMs possess distinct binding specificities. Until recently, the residues that are key to ligand binding in this family were unknown.…”

mentioning

confidence: 99%

Mutational Insights into the Roles of Amino Acid Residues in Ligand Binding for Two Closely Related Family 16 Carbohydrate Binding Modules

Agarwal

Dodd

et al. 2010

Carbohydrate binding modules (CBMs) are specialized proteins that bind to polysaccharides and oligosaccharides. Caldanaerobius polysaccharolyticus Man5ACBM16-1/CBM16-2 bind to glucose-, mannose-, and glucose/mannose-configured substrates. The crystal structures of the two proteins represent the only examples in CBM family 16, and studies that evaluate the roles of amino acid residues in ligand binding in this family are lacking. In this study, we probed the roles of amino acids (selected based on CBM16-1/ligand co-crystal structures) on substrate binding. Two tryptophan (Trp-20 and Trp-125) and two glutamine (Gln-81 and Gln-93) residues are shown to be critical in ligand binding. Additionally, several polar residues that flank the critical residues also contribute to ligand binding. The CBM16-1 Q121E mutation increased affinity for all substrates tested, whereas the Q21G and N97R mutants exhibited decreased substrate affinity. We solved CBM/substrate co-crystal structures to elucidate the molecular basis of the increased substrate binding by CBM16-1 Q121E. The Gln-121, Gln-21, and Asn-97 residues can be manipulated to fine-tune ligand binding by the Man5A CBMs. Surprisingly, none of the eight residues investigated was absolutely conserved in CBM family 16. Thus, the critical residues in the Man5A CBMs are either not essential for substrate binding in the other members of this family or the two CBMs are evolutionarily distinct from the members available in the current protein database. Man5A is dependent on its CBMs for robust activity, and insights from this study should serve to enhance our understanding of the interdependence of its catalytic and substrate binding modules.