title: Proteolysis of macrophage inflammatory protein-1alpha isoforms LD78beta and LD78alpha by neutrophil-derived serine proteases.
abstract: Macrophage inflammatory protein-1alpha (MIP-1alpha) is a chemokine that leads to leukocyte recruitment and activation at sites of infection. Controlling chemokine activity at sites of infection is important, since excess accumulation of leukocytes may contribute to localized tissue damage. Neutrophil-derived serine proteases modulate the bioactivity of chemokine and cytokine networks through proteolytic cleavage. Because MIP-1alpha is temporally expressed with neutrophils at sites of infection, we examined proteolysis of MIP-1alpha in vitro by the neutrophil-derived serine proteases: cathepsin G, elastase, and proteinase 3. Recombinant human MIP-1alpha isoforms LD78beta and LD78alpha were expressed and purified, and the protease cleavage sites were analyzed by mass spectrometry and peptide sequencing. Chemotactic activities of parent and cleavage molecules were also compared. Both LD78beta and LD78alpha were cleaved by neutrophil lysates at Thr16-Ser17, Phe24-Ile25, Tyr28-Phe29, and Thr31-Ser32. This degradation was inhibited by serine protease inhibitors phenylmethylsulfonyl fluoride and 4-(2-aminoethyl)-benzenesulfonyl fluoride. Incubation of the substrates with individual proteases revealed that cathepsin G preferentially cleaved at Phe24-Ile25 and Tyr28-Phe29, whereas elastase and proteinase 3 cleaved at Thr16-Ser17 and Thr31-Ser32. Proteolysis of LD78beta resulted in loss of chemotactic activity. The role of these proteases in LD78beta and LD78alpha degradation was confirmed by incubation with neutrophil lysates from Papillon-Lefevre syndrome patients, demonstrating that the cell lysates containing inactivated serine proteases could not degrade LD78beta and LD78alpha. These findings suggest that severe periodontal tissue destruction in Papillon-Lefevre syndrome may be related to excess accumulation of LD78beta and LD78alpha and dysregulation of the microbial-induced inflammatory response in the periodontium.
authors.affiliation: Human Craniofacial Genetics Section, NIDCR, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Ok Hee
authors.initials: OH
authors.lastname: Ryu
authors.forename: Sun Jin
authors.initials: SJ
authors.lastname: Choi
authors.forename: Erhan
authors.initials: E
authors.lastname: Firatli
authors.forename: Sung Won
authors.initials: SW
authors.lastname: Choi
authors.forename: P Suzanne
authors.initials: PS
authors.lastname: Hart
authors.forename: Rong-Fong
authors.initials: RF
authors.lastname: Shen
authors.forename: Guanghui
authors.initials: G
authors.lastname: Wang
authors.forename: Wells W
authors.initials: WW
authors.lastname: Wu
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Hart
mesh_terms: D000595:Amino Acid Sequence; D056649:Cathepsin G; D002403:Cathepsins / Q000737:chemistry / Q000378:metabolism; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D018925:Chemokines / Q000378:metabolism; D002633:Chemotaxis; D016207:Cytokines / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007249:Inflammation; D019402:Macrophage Inflammatory Proteins / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D053146:Myeloblastin; D009504:Neutrophils / Q000378:metabolism*; D010196:Pancreatic Elastase / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011480:Protease Inhibitors / Q000737:chemistry; D011487:Protein Conformation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013912:Threonine / Q000737:chemistry; D013997:Time Factors
chemical_list: D054405:Chemokine CCL3; D054407:Chemokine CCL4; D018925:Chemokines; D016207:Cytokines; D019402:Macrophage Inflammatory Proteins; D010455:Peptides; D011480:Protease Inhibitors; D020033:Protein Isoforms; D011994:Recombinant Proteins; D013912:Threonine; D012694:Serine; D010664:Phenylmethylsulfonyl Fluoride; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; D010196:Pancreatic Elastase; D053146:Myeloblastin