title: Characterization of a recombinant molecule covalently indistinguishable from human cerebroside-sulfate activator protein (CSAct or Saposin B).
abstract: Humans deficient in the cerebroside-sulfate activator protein (CSAct or Saposin B) are unable to catabolize sulfatide and other glycosphingolipids leading to their accumulation and neurodegenerative disease. Clinically this usually manifests as a form of metachromatic leukodystrophy (MLD). CSAct is a small water-soluble glycoprotein that apparently functions in the lysosome to solubilize sulfatide and other lipids enabling their interaction with soluble lysosomal hydrolases. CSAct activity can be measured in vitro by assay of its ability to activate sulfatide-sulfate hydrolysis by arylsulfatase A or ex vivo by its ability to functionally complement CSAct deficient fibroblast cell lines derived from MLD patients. A recombinant form of CSAct has been expressed in E. coli and processed in vitro to a form covalently indistinguishable from deglycosylated human CSAct isolated from human urine. Size-exclusion chromatography in combination with multi-angle laser-light scattering (SEC-MALLS) measurements demonstrate that both native and recombinant forms of the molecule behave as a dimer in the pH range 7.0-4.5. The CSAct activity assay showed that both recombinant and deglycosylated human urine CSAct efficiently activated sulfatide sulfate hydrolysis and provided functional complementation of CSAct-deficient cells. However, a D21N mutant form of recombinant CSAct could not functionally complement these cells despite full activity in the in vitro assay. It is concluded that while glycosylation is unnecessary for in vitro and ex vivo activity of CSAct, modification of the native N21 is necessary to prevent loss of ex vivo activity, possibly via protection from degradation.
authors.affiliation: The Pasarow Mass Spectrometry Laboratory, Department of Psychiatry and Biobehavioral Sciences, The Neuropsychiatric Institute, University of California, 405 Hilgard Ave., Los Angeles, CA 90095, USA. jpw@chem.ucla.edu
authors.forename: J P
authors.initials: JP
authors.lastname: Whitelegge
authors.forename: V
authors.initials: V
authors.lastname: Ahn
authors.forename: A J
authors.initials: AJ
authors.lastname: Norris
authors.forename: H
authors.initials: H
authors.lastname: Sung
authors.forename: A
authors.initials: A
authors.lastname: Waring
authors.forename: R L
authors.initials: RL
authors.lastname: Stevens
authors.forename: C B
authors.initials: CB
authors.lastname: Fluharty
authors.forename: G
authors.initials: G
authors.lastname: Prive
authors.forename: K F
authors.initials: KF
authors.lastname: Faull
authors.forename: A L
authors.initials: AL
authors.lastname: Fluharty
mesh_terms: D000626:Aminopeptidases / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002553:Cerebroside-Sulfatase / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003488:Cyanogen Bromide / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D005347:Fibroblasts / Q000201:enzymology / Q000378:metabolism; D015870:Gene Expression; D006023:Glycoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000172:deficiency; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry*; D012542:Scattering, Radiation; D021241:Spectrometry, Mass, Electrospray Ionization; D049128:Sphingolipid Activator Proteins; D013433:Sulfoglycosphingolipids / Q000378:metabolism; D013552:Swine
chemical_list: D004220:Disulfides; D006023:Glycoproteins; D011994:Recombinant Proteins; D049128:Sphingolipid Activator Proteins; D013433:Sulfoglycosphingolipids; D002553:Cerebroside-Sulfatase; D000626:Aminopeptidases; D003488:Cyanogen Bromide