title: Identification of new autolytic sites of recombinant truncated mature human fibroblast stromelysin by mass spectrometry.
abstract: Stromelysin has been proposed to play a major role in the pathologic degradation of diseased cartilage of osteoarthritis and rheumatoid arthritis patients. A truncated, recombinant form of this enzyme, with the sequence Phe83 to Thr260 (mSL-t), has been expressed and purified from E. coli to investigate its biochemical and biophysical properties, and to develop inhibitors for arthritis treatment. LC/ESI-MS technique was utilized for the characterization of mSL-t. The mass spectra of mSL-t showed the presence of a number of different protein components in addition to the full-length mSL-t form. We have demonstrated that protein degradation arose from autolysis. Molecular weights determined by LC/ESI-MS of these autolysis products allowed for the identification of new autolytic sites in mSL-t. Furthermore, two strategies were undertaken to prepare mSL-t free of degradation products. These include preparation of a mutant form of the enzyme in which Arg163 was substituted for Leu163 and purification of mSL-t using affinity chromatography. The LC/ESI-MS data of the mutant protein confirmed the Leu to Arg mutation. The affinity-purified material showed only one LC peak in the LC/MS chromatograms, and the mass spectrum of the peak identified only the intact protein, demonstrating that the full-length protein has been successfully separated from the autodegradation products and further autolysis of the enzyme has been prevented.
authors.affiliation: SAIC Frederick, NCI-FCRDC, Maryland, USA. ooronfle@ncifcrf.gov
authors.forename: M W
authors.initials: MW
authors.lastname: Qoronfleh
authors.forename: S K
authors.initials: SK
authors.lastname: Chowdhury
authors.forename: J
authors.initials: J
authors.lastname: Eshraghi
authors.forename: T
authors.initials: T
authors.lastname: Ho
authors.forename: P G
authors.initials: PG
authors.lastname: Brake
authors.forename: T
authors.initials: T
authors.lastname: Banks
authors.forename: J
authors.initials: J
authors.lastname: Huang
authors.forename: T
authors.initials: T
authors.lastname: Pulvino
authors.forename: B N
authors.initials: BN
authors.lastname: Jones
mesh_terms: D000595:Amino Acid Sequence; D002846:Chromatography, Affinity / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D006868:Hydrolysis; D013058:Mass Spectrometry / Q000379:methods; D019278:Matrix Metalloproteinase 3 / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
chemical_list: D011994:Recombinant Proteins; D019278:Matrix Metalloproteinase 3