title: Purification of a 75 kDa protein from the organelle matrix of human neutrophils and identification as N-acetylglucosamine-6-sulphatase.
abstract: A 75 kDa protein was purified to homogeneity from granule extracts of normal human granulocytes using Sephadex G-75 chromatography, Mono-S cation exchange chromatography and chromatofocusing. The protein consisted of one chain with a molecular mass of 75 kDa, as determined by SDS/PAGE. Tryptic peptide analysis by MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS and sequence analysis by MS/MS identified the protein to be N-acetylglucosamine-6-sulphatase (EC 3.1.6.14). The identity of the protein was confirmed by demostrating enzymatic activity towards the substrate N-acetylglucosamine 6-sulphate. The enzyme was active over a broad pH range with an optimum of pH 7.0, and showed a K(m) value of 13.0 mM and a V(max) value of approximately 1.8 microM/min per mg. The enzyme also showed O-desulphation activity towards heparan sulphate-derived saccharides. Subcellular fractionation of neutrophil organelles showed the presence of enzymatic activity mainly in the same fractions as primary granules. Furthermore, PMA treatment of the neutrophils induced release of the enzyme, indicating its matrix protein nature. The presence of N-acetylglucosamine-6-sulphatase in human neutrophils implies that neutrophils may play a role in the modulation of cell surface molecules and extracellular matrix by O-desulphation.
authors.affiliation: Department of Medical Sciences, Clinical Chemistry, Uppsala University, SE-751 85, Uppsala, Sweden. shengyuan.xu@medsci.uu.se
authors.forename: Shengyuan
authors.initials: S
authors.lastname: Xu
authors.forename: Linshu
authors.initials: L
authors.lastname: Zhao
authors.forename: Anders
authors.initials: A
authors.lastname: Larsson
authors.forename: Emanuel
authors.initials: E
authors.lastname: Smeds
authors.forename: Marion
authors.initials: M
authors.lastname: Kusche-Gullberg
authors.forename: Per
authors.initials: P
authors.lastname: Venge
mesh_terms: D006801:Humans; D009504:Neutrophils / Q000737:chemistry*; D015388:Organelles / Q000737:chemistry*; D013379:Substrate Specificity; D013429:Sulfatases / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D013429:Sulfatases; C018719:N-acetylglucosamine-6-sulfatase