title: Interconversion of the specificities of human lysosomal enzymes associated with Fabry and Schindler diseases.
abstract: The human lysosomal enzymes alpha-galactosidase (alpha-GAL, EC 3.2.1.22) and alpha-N-acetylgalactosaminidase (alpha-NAGAL, EC 3.2.1.49) share 46% amino acid sequence identity and have similar folds. The active sites of the two enzymes share 11 of 13 amino acids, differing only where they interact with the 2-position of the substrates. Using a rational protein engineering approach, we interconverted the enzymatic specificity of alpha- GAL and alpha-NAGAL. The engineered alpha-GAL (which we call alpha-GAL(SA)) retains the antigenicity of alpha-GAL but has acquired the enzymatic specificity of alpha-NAGAL. Conversely, the engineered alpha-NAGAL (which we call alpha-NAGAL(EL)) retains the antigenicity of alpha-NAGAL but has acquired the enzymatic specificity of the alpha-GAL enzyme. Comparison of the crystal structures of the designed enzyme alpha-GAL(SA) to the wild-type enzymes shows that active sites of alpha-GAL(SA) and alpha-NAGAL superimpose well, indicating success of the rational design. The designed enzymes might be useful as non-immunogenic alternatives in enzyme replacement therapy for treatment of lysosomal storage disorders such as Fabry disease.
authors.affiliation: Departments of Biochemistry & Molecular Biology, University of Massachusetts, Amherst, Massachusetts 01003, USA.
authors.forename: Ivan B
authors.initials: IB
authors.lastname: Tomasic
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Metcalf
authors.forename: Abigail I
authors.initials: AI
authors.lastname: Guce
authors.forename: Nathaniel E
authors.initials: NE
authors.lastname: Clark
authors.forename: Scott C
authors.initials: SC
authors.lastname: Garman
mesh_terms: D000595:Amino Acid Sequence; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000737:chemistry; D018360:Crystallography, X-Ray / Q000379:methods; D000795:Fabry Disease / Q000201:enzymology*; D006023:Glycoproteins / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D016464:Lysosomal Storage Diseases / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D017510:Protein Folding; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D000519:alpha-Galactosidase / Q000737:chemistry*; D048809:alpha-N-Acetylgalactosaminidase / Q000737:chemistry*
chemical_list: D002241:Carbohydrates; D006023:Glycoproteins; D000519:alpha-Galactosidase; D048809:alpha-N-Acetylgalactosaminidase
references.citation: Angew Chem Int Ed Engl. 2007;46(18):3212-36
references.pmid: 17450624
references.citation: Pediatrics. 2005 Mar;115(3):e344-55
references.pmid: 15713906
references.citation: Nucleic Acids Res. 2009 Jan;37(Database issue):D233-8
references.pmid: 18838391
references.citation: Genet Med. 2008 May;10(5):353-8
references.pmid: 18496035
references.citation: J Mol Biol. 2004 May 28;339(2):413-22
references.pmid: 15136043
references.citation: J Mol Biol. 2009 Oct 23;393(2):435-47
references.pmid: 19683538
references.citation: Mol Genet Metab. 2008 Jul;94(3):319-25
references.pmid: 18424138
references.citation: N Engl J Med. 2001 Jul 5;345(1):9-16
references.pmid: 11439963
references.citation: Nephrol Dial Transplant. 2006 Feb;21(2):345-54
references.pmid: 16204287
references.citation: Mol Genet Metab. 2007 Feb;90(2):157-63
references.pmid: 17079176
references.citation: Am J Hum Genet. 2009 Nov;85(5):569-80
references.pmid: 19853240
references.citation: J Biol Chem. 2003 May 30;278(22):20313-8
references.pmid: 12657636
references.citation: J Biol Chem. 2010 Feb 5;285(6):3625-3632
references.pmid: 19940122
references.citation: Biochemistry. 1995 Feb 7;34(5):1489-99
references.pmid: 7849008
references.citation: Mol Genet Metab. 2009 Jan;96(1):4-12
references.pmid: 19022694
references.citation: Structure. 2002 Mar;10(3):425-34
references.pmid: 12005440
references.citation: Clin Chim Acta. 1981 May 5;112(2):247-51
references.pmid: 6263521
references.citation: Structure. 1997 Dec 15;5(12):1557-69
references.pmid: 9438862
references.citation: J Biol Chem. 1990 Dec 15;265(35):21859-66
references.pmid: 2174888
references.citation: Acta Crystallogr A. 1991 Mar 1;47 ( Pt 2):110-9
references.pmid: 2025413
references.citation: J Mol Biol. 2004 Mar 19;337(2):319-35
references.pmid: 15003450
references.citation: JAMA. 2001 Jun 6;285(21):2743-9
references.pmid: 11386930
references.citation: Am J Hum Genet. 2006 Jul;79(1):31-40
references.pmid: 16773563