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BMC Biochem
2017 May 25;181:9. doi: 10.1186/s12858-017-0085-1.
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Chemical and structural characterization of α-N-acetylgalactosaminidase I and II from starfish, asterina amurensis.
Rashid MH
,
Sadik G
,
Alam AK
,
Tanaka T
.
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BACKGROUND: The marine invertebrate starfish was found to contain a novel α-N-acetylgalactosaminidase, α-GalNAcase II, which catalyzes removal of terminal α-N-acetylgalactosamine (α-GalNAc), in addition to a typical α-N-acetylgalactosaminidase, α-GalNAcase I, which catalyzes removal of terminal α-N-acetylgalactosamine (α-GalNAc) and, to a lesser extent, galactose. The interrelationship between α-GalNAcase I and α-GalNAcase II and the molecular basis of their differences in substrate specificity remain unknown.
RESULTS: Chemical and structural comparisons between α-GalNAcase I and II using immunostaining, N-terminal amino acid sequencing and peptide analysis showed high homology to each other and also to other glycoside hydrolase family (GHF) 27 members. The amino acid sequence of peptides showed conserved residues at the active site as seen in typical α-GalNAcase. Some substitutions of conserved amino acid residues were found in α-GalNAcase II that were located near catalytic site. Among them G171 and A173, in place of C171 and W173, respectively in α-GalNAcase were identified to be responsible for lacking intrinsic α-galactosidase activity of α-GalNAcase II. Chemical modifications supported the presence of serine, aspartate and tryptophan as active site residues. Two tryptophan residues (W16 and W173) were involved in α-galactosidase activity, and one (W16) of them was involved in α-GalNAcase activity.
CONCLUSIONS: The results suggested that α-GalNAcase I and II are closely related with respect to primary and higher order structure and that their structural differences are responsible for difference in substrate specificities.
Fig. 1. Deglycosylation and immunostaining of α-GalNAcase I and II. a SDS-PAGE of purified and deglycosylated α-GalNAcase I and II. The proteins were separated on SDS-PAGE and the gel was stained with CBB R-250 solution. S, Molecular weight marker; −, purified enzyme; +, enzyme treated with PNGase F. b Immunostaining between α-GalNAcase and anti-α-GalNAcase antibodies. 1, Antiserum to human α-GalNAcase; 2, antiserum to squid α-GalNAcase I; and 3, antiserum to squid α-GalNAcase II. Lane I, Enzyme α-GalNAcase I; and Lane II, Enzyme α-GalNAcase II
Fig. 2. Peptide maps of α-GalNAcase I and II. RP-HPLC separation of lysylendopeptidase digested peptides of α-GalNAcase I a and α-GalNAcase-II b. Blank sample (without enzyme) contain the peaks b. The peptide peaks indicated with asterisk are subjected for amino acid sequence by MS/MS analysis. See text for details
Fig. 3. MS/MS analysis for amino acid sequence of the peptides of α-GalNAcase I and II. The individually obtained peptide fraction from RP-HPLC were subjected to MS/MS fragmentation. Amino acid sequences of peptides were determined from the fragmentation pattern by Bruker Data Analysis software (version 4.0). a, Peptide 17 of enzyme I; b, peptide 15 of enzyme I; c, peptide 13 of enzyme II; d peptide 16 of enzyme II
Fig. 4. Summary of peptide sequences of α-GalNAcase I and II from starfish and alignment with that of chicken α-GalNAcases. The peptide sequences α-GalNAcase I and II were aligned with the corresponding sequence from chicken (Ch) α-GalNAcase. The underlined number indicated the respective peptide on Fig. 2. The peptide sequences marked with asterisk were analyzed by sequence analyzer. Numbering of amino acid started from N-terminal leucine of chicken α-GalNAcase. (Blank), means not determined the sequence. (.), means absent of amino acid residue. The conserved active site residues are shown in red. Boxed peptides comprise the N-acetyl recognition loop. The substitution of conserved residues in α-GalNAcase II as compared to typical α-GalNAcase are shown in green. Asp, Acremonium species α-GalNAcase (Ashida et al. [15]); Hu-α-Gal, Human α-galactosidase (Tsuji et al. [24])
Fig. 5. Effects of tryptophan modification and various monosaccharides on α-GalNAcase and α-galactosidase activities of α-GalNAcase I. a The NBS titration curve of percent residual activity versus the number of tryptophan residues modified was poltted for determination of number of tryptophan residues involved in α-GalNAcase and α-galactosidase activities of α-GalNAcase I. b Effect of various monosaccharide on α-GalNAcase I. α-GalNAcase I was preincubated with various monosaccharide (Man, Gal, Glc, GalNAc and GlcNAc) at 10 mM concentration for 30 min at 25 °C and determined the residual α-galactosidase (□) and α-GalNAcase (■) activities. Data are presented as mean value of three determinations
Fig. 6. Double displacement catalytic mechanisms of starfish α-GalNAcase I and II. Step 1: D140 from α-GalNAcase makes a nucleophilic attack on C1 of terminal α-GalNAc of substrate, cleaving the glycosidic linkage and producing a covalent intermediate complex. Step 2: A water molecule, deprotonated by D201, makes a nucleophilic attack on C1 of α-GalNAc and cleave the covalent enzyme-substrate intermediate complex and release N-acetylgalactosamine
Fig. 7. Proposed interactions between ligand and active site residues of α-GalNAcase I and II. The α-GalNAc ligand was placed on the center of active site and the interactions between ligand and active site residues were analyzed by software ACDLabs Free software 5.0. The ligand is surrounded by amino acid side chains labeled in the active site of α-GalNAcase I a and α-GalNAcase II b. The hydrogen bond and polar interactions are shown in red and hydrophobic interactions are shown in blue
Ashida,
Molecular cloning of cDNA encoding alpha-N-acetylgalactosaminidase from Acremonium sp. and its expression in yeast.
2000, Pubmed
Ashida,
Molecular cloning of cDNA encoding alpha-N-acetylgalactosaminidase from Acremonium sp. and its expression in yeast.
2000,
Pubmed
Calcutt,
Identification, molecular cloning and expression of an alpha-N-acetylgalactosaminidase gene from Clostridium perfringens.
2002,
Pubmed
Chabás,
A new infantile case of alpha-N-acetylgalactosaminidase deficiency. Cardiomyopathy as a presenting symptom.
2007,
Pubmed
Conzelmann,
Glycolipid and glycoprotein degradation.
1987,
Pubmed
Dean,
The identification of alpha-galactosidase B from human liver as an alpha-N-acetylgalactosaminidase.
1977,
Pubmed
Diezel,
An improved procedure for protein staining in polyacrylamide gels with a new type of Coomassie Brilliant Blue.
1972,
Pubmed
Garman,
The 1.9 A structure of alpha-N-acetylgalactosaminidase: molecular basis of glycosidase deficiency diseases.
2002,
Pubmed
Garman,
The molecular defect leading to Fabry disease: structure of human alpha-galactosidase.
2004,
Pubmed
Gerhard,
The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).
2004,
Pubmed
Harhay,
Characterization of 954 bovine full-CDS cDNA sequences.
2005,
Pubmed
Harun-Or-Rashid,
Purification and characterization of alpha-N-acetylgalactosaminidases I and II from the starfish Asterina amurensis.
2010,
Pubmed
,
Echinobase
Henrissat,
Structural and sequence-based classification of glycoside hydrolases.
1997,
Pubmed
Hiraiwa,
Human placental sialidase: further purification and characterization.
1988,
Pubmed
Hujová,
Characterization of gana-1, a Caenorhabditis elegans gene encoding a single ortholog of vertebrate alpha-galactosidase and alpha-N-acetylgalactosaminidase.
2005,
Pubmed
Itoh,
alpha-N-Acetylgalactosaminidase from squid liver: purification and characterization of two enzymes.
1984,
Pubmed
Kanzaki,
Novel lysosomal glycoaminoacid storage disease with angiokeratoma corporis diffusum.
1989,
Pubmed
Kestwal,
Purification of beta-galactosidase from Erythrina indica: involvement of tryptophan in active site.
2007,
Pubmed
Kodama,
A new case of alpha-N-acetylgalactosaminidase deficiency with angiokeratoma corporis diffusum, with Ménière's syndrome and without mental retardation.
2001,
Pubmed
Kusiak,
Purification and properties of the two major isozymes of alpha-galactosidase from human placenta.
1978,
Pubmed
Laemmli,
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
1970,
Pubmed
Levy,
Purification and properties of alpha-N-acetylgalactosaminidase from Clostridium perfringens.
1980,
Pubmed
Liu,
Bacterial glycosidases for the production of universal red blood cells.
2007,
Pubmed
Nakagawa,
Purification and characterization of alpha-N-acetylgalactosaminidase from skipjack liver.
1987,
Pubmed
Sadik,
Chemical and immunological characterization of the two alpha-N-acetylgalactosaminidases from squid liver.
2009,
Pubmed
Schindler,
Neuroaxonal dystrophy due to lysosomal alpha-N-acetylgalactosaminidase deficiency.
1989,
Pubmed
Smith,
Measurement of protein using bicinchoninic acid.
1985,
Pubmed
Spande,
The reactivity toward N-bromosuccinimide of tryptophan in enzymes, zymogens, and inhibited enzymes.
1966,
Pubmed
Sung,
Purification and partial characterization of porcine liver alpha-N-acetylgalactosaminidase.
1980,
Pubmed
Tsuji,
Molecular cloning of a full-length cDNA for human alpha-N-acetylgalactosaminidase (alpha-galactosidase B).
1989,
Pubmed
Tsuji,
Signal sequence and DNA-mediated expression of human lysosomal alpha-galactosidase A.
1987,
Pubmed
Uda,
alpha-N-acetylgalactosaminidase from the limpet, Patella vulgata.
1977,
Pubmed
van Diggelen,
Lysosomal alpha-N-acetylgalactosaminidase deficiency: a new inherited metabolic disease.
1987,
Pubmed
Vasella,
Glycosidase mechanisms.
2002,
Pubmed
Zhu,
Cloning and characterization of a cDNA encoding chicken liver alpha-N-acetylgalactosaminidase.
1993,
Pubmed
