Interconversion of the specificities of human lysosomal enzymes associated with Fabry and Schindler diseases.
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.
Tomasic
Ivan B
IB
Departments of Biochemistry & Molecular Biology, University of Massachusetts, Amherst, Massachusetts 01003, USA.
Metcalf
Matthew C
MC
Guce
Abigail I
AI
Clark
Nathaniel E
NE
Garman
Scott C
SC
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*
D002241:Carbohydrates; D006023:Glycoproteins; D000519:alpha-Galactosidase; D048809:alpha-N-Acetylgalactosaminidase
Angew Chem Int Ed Engl. 2007;46(18):3212-36
17450624
Pediatrics. 2005 Mar;115(3):e344-55
15713906
Nucleic Acids Res. 2009 Jan;37(Database issue):D233-8
18838391
Genet Med. 2008 May;10(5):353-8
18496035
J Mol Biol. 2004 May 28;339(2):413-22
15136043
J Mol Biol. 2009 Oct 23;393(2):435-47
19683538
Mol Genet Metab. 2008 Jul;94(3):319-25
18424138
N Engl J Med. 2001 Jul 5;345(1):9-16
11439963
Nephrol Dial Transplant. 2006 Feb;21(2):345-54
16204287
Mol Genet Metab. 2007 Feb;90(2):157-63
17079176
Am J Hum Genet. 2009 Nov;85(5):569-80
19853240
J Biol Chem. 2003 May 30;278(22):20313-8
12657636
J Biol Chem. 2010 Feb 5;285(6):3625-3632
19940122
Biochemistry. 1995 Feb 7;34(5):1489-99
7849008
Mol Genet Metab. 2009 Jan;96(1):4-12
19022694
Structure. 2002 Mar;10(3):425-34
12005440
Clin Chim Acta. 1981 May 5;112(2):247-51
6263521
Structure. 1997 Dec 15;5(12):1557-69
9438862
J Biol Chem. 1990 Dec 15;265(35):21859-66
2174888
Acta Crystallogr A. 1991 Mar 1;47 ( Pt 2):110-9
2025413
J Mol Biol. 2004 Mar 19;337(2):319-35
15003450
JAMA. 2001 Jun 6;285(21):2743-9
11386930
Am J Hum Genet. 2006 Jul;79(1):31-40
16773563