Cloning, sequence, expression and characterization of human β-mannosidase

• Authors: Zahoor Qadir Samra , Muhammad Amin Athar
• Languages of publication: EN

Abstracts

EN

β-mannosidase (EC 3.2.1.25, MANB) dissects the non-reducing end of N-linked mannose moieties of glycoproteins in eukaryotic cells. The human β-mannosidase gene was amplified by RT-PCR, cloned and sequenced. The DNA sequence was compared with reported human β-mannosidase DNA sequence and sixteen nucleotide differences were found. The deduced amino-acid sequence showed that seven codons coded the same amino acids and nine codons coded different amino acids with reference to nucleotide substitution positions but did not affect recombinant MANB enzyme activity. No splice mutation was observed after comparison with reported MANB DNA sequences. A 75% homology of deduced amino-acid sequence was observed with mouse, goat and bovine β-mannosidase amino-acid sequences. The cloned β-mannosidase gene was subcloned into pET22b+ and pET28a+ expression vectors to transform the BL21-codon plus cells for expression of recombinant MAN22 and MAN28 enzymes, respectively. The optimized conditions for overexpression of recombinant β-mannosidase enzyme were induction with 1 mM IPTG for 12 h at 37°C. The expressed β-mannosidase enzyme was purified to homogeneity by a combination of DEAE-ion exchange and size exclusion chromatography. The molecular mass of MAN22 and MAN28 enzymes is 97 kDa by SDS/PAGE and is confirmed by western blot analysis. The recombinant enzymes are active at 37°C and at pH 5.0 and showed activity with p-nitrophenyl-β-d-mannopyranoside and not with p-nitrophenyl-α-d-mannopyranoside. The Km value of enzymes was 2.53 mM. The enzyme activity was inhibited by Zn2+, Co2+, Cu2+, Pb2+, Ag1+, iodoacetate, SDS, DMF, DMSO and ethanol. Fe3+, Ca2+ Mg2+, Mn2+, Triton X-100 and PMSF did not inhibit the enzyme activity. Northern blot analysis showed a transcript of about 3.7 kb in all cells and tissues studied. This is the first report on the expression and characterization of recombinant human MANB enzyme.

Keywords

EN

immunochemical reactions; southern and northern analysis; β-mannosidase; sequence; expression; cloning

• Publisher: Polish Biochemical Society
• Journal: Acta Biochimica Polonica
• Year: 2008
• Volume: 55
• Issue: 3
• Pages: 479-490

Dates

published

2008

received

2008-02-12

revised

2008-08-20

accepted

2008-09-16

(unknown)

2008-09-17

Contributors

author

Zahoor Qadir Samra

• Institute of Biochemistry and Biotechnology, Quaid-i-Azam Campus, University of the Punjab, Lahore, Pakistan

author

Muhammad Amin Athar

• Institute of Biochemistry and Biotechnology, Quaid-i-Azam Campus, University of the Punjab, Lahore, Pakistan

References

• Alkhayat AH, Kraemer SA, Leipprandt JR, Macek M, Kleijer WJ, Friderici KH (1998) Human β-mannosidase cDNA characterization and first identification of a mutation associated with human β-mannosidosis. Hum Mol Genet 7: 75-83.
• Ademark P, Lundqvist J, Hagglund P, Tenkanen M, Torto N, Tjerneld F, Stalbrand H (1999) Hydrolytic properties of a β-mannosidase purified from Aspergillus niger. J Biotechnol 75: 281-289.
• Abbitt B, Jones MZ, Kasari TR, Storts RW, Templeton JW, Holland PS, Castenson PE (1991) Β-mannosidosis in twelve Salers calves. J Am Vet Med Assoc 198: 109-113.
• Ausubel FM, Brent R, Kingston RE, More DD, Seidman JG, Smith JA, Struhl K (2002) Short protocols in molecular biology, 1st edn. John Wiley & Sons Inc, Canada.
• Bauer MW, Bylina EJ, Swanson RV, Kelly RM (1996) Comparison of a β-glucosidase and a β-mannosidase from the hyperthermophilic archaeon Pyrococcus furiosus. Purification, characterization, gene cloning, and sequence analysis. J Biol Chem 271: 23749-23755.
• Beccari T, Bibi L, Stinchi S, Stirling JL, Orlacchio A (2001) Mouse β-mannosidase: cDNA cloning, expression, and chromosomal localization. Biosci Rep 21: 315-323.
• Bedilu R, Nummy KA, Cooper A, Wevers R, Smeitink J, Kleijer WJ, Friderici KH (2002) Variable clinical presentation of lysosomal β-mannosidosis in patients with null mutations. Mol Genet Metab 77: 282-290.
• Bradford MM (1976) A dye binding assay for protein. Anal Biochem 72: 248-254.
• Bryan L, Schmutz S, Hodges SD, Snyder FF (1990) Bovine β-mannosidase deficiency. Biochem Biophys Res Commun 173: 491-495.
• Chen H, Leipprandt JR, Traviss CE, Sopher BL, Jones MZ, Cavanagh KT, Friderici KH (1995) Molecular cloning and characterization of bovine β-mannosidase. J Biol Chem 270: 3841-3848.
• Cooper A, Hatton C, Thornley M, Sardharwalla IB (1988) Human β-mannosidase deficiency: biochemical findings in plasma, fibroblasts, white cells and urine. J Inherit Metab Dis 11: 17-29.
• Cooper A, Wraith JE, Savage WJ, Thornley M, Noronha MJ (1991) β-mannosidase deficiency in a female infant with epileptic encephalopathy. J Inherit Metab Dis 14: 18-22.
• Duffaud GD, McCutchen CM, Leduc P, Parker KN, Kelly RM (1997) Purification and characterization of extremely thermostable β-mannanase, β-mannosidase, and alpha-galactosidase from the hyperthermophilic eubacterium Thermotoga neapolitana 5068. Appl Environ Microbiol 63: 169-177.
• Gort L, Duque J, Fabeiro JM, Zulaica A, Coll MJ, Chabas A (2006) Molecular analysis in two β-mannosidosis patients: description of a new adult case. Mol Genet Metab 89: 398-400.
• Guadalupi R, Bernard M, Orlacchio A, Foglietti MJ, Emiliani C (1996) Purification and properties of human urinary β-d-mannosidase. Biochim Biophys Acta 1293: 9-16.
• Harlow ED, Lane D (1988) Antibodies, A laboratory manual. Cold Spring Harbour Labs., USA.
• Johnstone A, Thrope R (1982) Immunochemistry in practice. 2nd edn. Blackwell Scientific Publication, UK.
• Jolly RD, Thompson KG, Bayliss SL, Vidler BM, Orr MB, Healy PJ (1990) β-Mannosidosis in a Salers calf: a new storage disease of cattle. N Z Vet J 38: 102-105.
• Jones MZ, Cunningham JG, Dade AW, Alessi DM, Mostosky UV, Vorro JR, Benitez JT, Lovell KL (1983) Caprine β-mannosidosis: clinical and pathological features. J Neuropathol Exp Neurol 42: 268-285.
• Jones MZ, Rathke EJ, Gage DA, Costello CE, Murakami K, Ohta M, Matsuura F (1992) Oligosaccharides accumulated in the bovine β-mannosidosis kidney. J Inherit Metab Dis 15: 57-67.
• Kleijer WJ, Hu P, Thoomes R, Boer M, Huijmans JG, Blom W, Van Diggelen OP, Seemanova E, Macek M (1990) Β-mannosidase deficiency: heterogeneous manifestation in the first female patient and her brother. J Inherit Metab Dis 13: 867-872.
• Kozak M (1984) Compilation and analysis of sequence upstream from the translation start site in eukaryotic mRNA. Nucleic Acid Res 12: 857-872.
• Kurakake M, Komaki T (2001) Production of β-mannanase and β-mannosidase from Aspergillus awamori K4 and their properties. Curr Microbiol 42: 377-380.
• Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.
• Leipprandt JR, Kraemer SA, Haithcock BE, Chen H, Dyme JL, Cavanagh KT, Friderici KH, Jones MZ (1996) Caprine β-mannosidase: sequencing and characterization of the cDNA and identification of the molecular defect of caprine β-mannosidosis. Genomics 37: 51-56.
• Levade T, Graber D, Flurin V, Delisle MB, Pieraggi MT, Testut MF, Carriere JP, Salvayre R (1994) Human β-mannosidase deficiency associated with peripheral neuropathy. Ann Neurol 35: 116-119.
• Lovell KL, Kranich RJ, Cavanagh KT (1994) Biochemical and histochemical analysis of lysosomal enzyme activities in caprine β-mannosidosis. Mol Chem Neuropathol 21: 61-74.
• Mo B, Bewley JD (2002) Β-mannosidase activity during and following germination of tomato (Lycopersicon esculentum Mill.) seeds. Purification, cloning and characterization. Planta 215: 141-152.
• Patterson JS, Jones MZ, Lovell KL, Abbitt B (1991) Neuropathology of bovine β-mannosidosis. J Neuropathol Exp Neurol 50: 538-546.
• Pearce RD, Callahan JW, Novak A, Little PB, Clarke JT (1990) Properties of partially purified goat kidney β-d-mannosidase. Br Vet J 146: 270-280.
• Percheron F, Foglietti MJ, Bernard M, Ricard B (1992) Mammalian β-d-mannosidase and β-mannosidosis. Biochimie 74: 5-11.
• Poenaru L, Akli S, Rocchiccioli F, Eydoux P, Zamet P (1992) Human β-mannosidosis: a 3-year-old boy with speech impairment and emotional instability. Clin Genet 41: 331-334.
• Plummer DT (1987) An introduction to practical biochemistry. 3rd edn, McGraw Hill, UK.
• Render JA, Lovell KL, Jones MZ (1988) Otic pathology of caprine β-mannosidosis. Vet Pathol 25: 437-442.
• Rodriguez-Serna M, Botella-Estrada R, Chabas A, Col MJ, Oliver V, Febrer MI, Aliaga A (1996) Angiokeratoma corporis diffusum associated with β-mannosidase deficiency. Arch Dermatol 132: 1219-1222.
• Sambrook J, Russel DW (2001) Molecular cloning, A laboratory manual. 3rd edn. Cold Spring Harbour Labs, USA.
• Samra ZQ, Athar MA (2008) Production and immunocomparative study of human lysosomal β-mannosidase antibodies. Pak J Zool 40: 185-192.
• Sedel F, Friderici K, Nummy K, Caillaud C, Chabli A, Durr A, Lubetzki C, Agid Y (2006) Atypical Gilles de la Tourette syndrome with β-mannosidase deficiency. Arch Neurol 63: 129-131.
• Sopher BL, Traviss CE, Cavanagh KT, Jones MZ, Friderici KH (1993) Bovine kidney β-mannosidase: purification and characterization. Biochem J 289: 343-347.
• Thiel T, Bissen S, Lyons EM (2002) Biotechnology, DNA to Protein, 1st edn, McGraw Hill companies, Inc, NY, USA.
• Towbin HK, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose membrane. Proc Natl Acad Sci USA 76: 4350-4359.
• Uchino Y, Fukushige T, Yotsumoto S, Hashiguchi T, Taguchi H, Suzuki N, Konohana I, Kanzak T (2003) Morphological and biochemical studies of human β-mannosidosis: identification of a novel β-mannosidase gene mutation. Br J Dermatol 149: 23-29.
• van Pelt J, Dorland L, Duran M, Hokke CH, Kamerling JP, Vliegenthart JF (1990) Sialyl-alpha 2-6-mannosyl-β 1-4-N-acetylglucosamine, a novel compound occurring in urine of patients with β-mannosidosis. J Biol Chem 265: 19685-19689.
• von Heijne G (1986) A new method for predicting signal sequence cleavage site. Nucleic Acids Res 14: 4683-4690.
• Wysocki LJ, Sato VL (1978) Panning for lymphocytes: A method for cell selection. Proc Natl Acad Sci USA 75: 2844-2848.