title: A novel single-chain antibody fragment for detection of mannose 6-phosphate-containing proteins: application in mucolipidosis type II patients and mice.
abstract: Newly synthesized soluble lysosomal hydrolases require mannose 6-phosphate (Man6P) residues on their oligosaccharides for their transport to lysosomes. The formation of Man6P residues is catalyzed by the GlcNAc-1-phosphotransferase, which is defective in the lysosomal storage disorders mucolipidosis type II (ML II) and ML III. Both hypersecretion and reduced intracellular level of lysosomal enzymes as well as direct sequencing of GlcNAc-1-phosphotransferase genes are important diagnostic markers for ML II and ML III. A high-affinity Man6P-specific single-chain antibody fragment was generated, allowing the rapid indirect demonstration of defective GlcNAc-1-phosphotransferase. In media and extracts of cultured fibroblasts of healthy controls but not of ML II and ML III patients, several Man6P-containing proteins could be detected by anti-Man6P Western blotting. Immunoprecipitation of Man6P-containing proteins from conditioned media or mouse brain extracts followed by arylsulfatase A and cathepsin D Western blotting confirmed the specificity of the antibody fragment for lysosomal proteins. Application of the antibody fragment in immunohistochemistry of human brain slices from nonaffected patients showed strong neuronal immunoreactivity, which was not observed in cortical sections of an ML II patient. Finally, in brain extracts of a novel GlcNAc-1-phosphotransferase knock-in mouse no Man6P-containing proteins were detectable. Thus, the single-chain antibody fragment against Man6P was demonstrated to allow the specific, rapid, and convenient detection of Man6P-containing proteins and facilitates the diagnosis of ML II and ML III.
authors.affiliation: University Medical Center Hamburg-Eppendorf, Department of Biochemistry, Children's Hospital, Martinistr. 52, Bld N27, 20246 Hamburg, Germany.
authors.forename: Sven
authors.initials: S
authors.lastname: Müller-Loennies
authors.forename: Giovanna
authors.initials: G
authors.lastname: Galliciotti
authors.forename: Katrin
authors.initials: K
authors.lastname: Kollmann
authors.forename: Markus
authors.initials: M
authors.lastname: Glatzel
authors.forename: Thomas
authors.initials: T
authors.lastname: Braulke
mesh_terms: D000818:Animals; D002675:Child, Preschool; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D055879:Gene Knock-In Techniques; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000378:metabolism*; D008297:Male; D008360:Mannosephosphates / Q000378:metabolism*; D051379:Mice; D008875:Middle Aged; D009081:Mucolipidoses / Q000175:diagnosis* / Q000201:enzymology; D011506:Proteins / Q000737:chemistry*; D057127:Single-Chain Antibodies / Q000235:genetics / Q000378:metabolism*; D017855:Transferases (Other Substituted Phosphate Groups) / Q000235:genetics / Q000378:metabolism
chemical_list: D007128:Immunoglobulin Fragments; D008360:Mannosephosphates; D011506:Proteins; D057127:Single-Chain Antibodies; C027693:mannose-6-phosphate; D017855:Transferases (Other Substituted Phosphate Groups); C548056:GNPTAB protein, mouse
references.citation: Biochim Biophys Acta. 2009 Apr;1793(4):605-14
references.pmid: 19046998
references.citation: Birth Defects Orig Artic Ser. 1975;11(6):307-10
references.pmid: 127632
references.citation: J Biol Chem. 1991 Nov 15;266(32):21874-9
references.pmid: 1939211
references.citation: J Biol Chem. 1990 Apr 25;265(12):6650-5
references.pmid: 1969863
references.citation: Hum Gene Ther. 2008 Aug;19(8):857; author reply 858
references.pmid: 18754711
references.citation: J Biol Chem. 2005 Oct 28;280(43):36141-9
references.pmid: 16120602
references.citation: Proteomics. 2002 Aug;2(8):1026-40
references.pmid: 12203898
references.citation: Glycobiology. 2008 Sep;18(9):664-78
references.pmid: 18621992
references.citation: Nat Med. 2005 Oct;11(10):1109-12
references.pmid: 16200072
references.citation: EMBO J. 2004 May 19;23(10):2019-28
references.pmid: 15085180
references.citation: J Hum Genet. 2009 Mar;54(3):145-51
references.pmid: 19197337
references.citation: Biochim Biophys Acta. 2009 Apr;1793(4):684-96
references.pmid: 19111581
references.citation: Hum Mutat. 2005 Oct;26(4):308-14
references.pmid: 16116615
references.citation: J Cell Biol. 1987 Jun;104(6):1735-42
references.pmid: 2953746
references.citation: J Biol Chem. 1989 May 15;264(14):7970-5
references.pmid: 2542255
references.citation: Proc Natl Acad Sci U S A. 1997 Oct 28;94(22):11963-8
references.pmid: 9342345
references.citation: Biochem J. 2003 Jan 1;369(Pt 1):55-62
references.pmid: 12350228
references.citation: Humangenetik. 1970;9(2):113-39
references.pmid: 4246487
references.citation: Am J Hum Genet. 2006 Mar;78(3):451-63
references.pmid: 16465621
references.citation: J Med Genet. 2004 Apr;41(4):e52
references.pmid: 15060128
references.citation: Methods Enzymol. 2000;312:205-16
references.pmid: 11070874
references.citation: J Clin Invest. 2000 Mar;105(5):673-81
references.pmid: 10712439
references.citation: Biol Chem. 2009 Jul;390(7):521-7
references.pmid: 19426136
references.citation: Hum Mutat. 2004 Dec;24(6):535
references.pmid: 15532026
references.citation: J Biol Chem. 1997 Feb 21;272(8):4804-13
references.pmid: 9030536
references.citation: Biochim Biophys Acta. 2009 Apr;1793(4):625-35
references.pmid: 18977398
references.citation: Birth Defects Orig Artic Ser. 1975;11(6):295-9
references.pmid: 811281
references.citation: Mol Genet Metab. 2006 Aug;88(4):359-63
references.pmid: 16630736
references.citation: J Clin Invest. 1981 May;67(5):1574-9
references.pmid: 6262380
references.citation: Biochem Biophys Res Commun. 1981 Feb 12;98(3):761-7
references.pmid: 6452876
references.citation: Mol Cell Proteomics. 2008 Jan;7(1):58-70
references.pmid: 17848585
references.citation: Am J Physiol Endocrinol Metab. 2001 Dec;281(6):E1221-9
references.pmid: 11701437