Production and Characterization of Recombinant Active Mouse Gelatinase B from Eukaryotic Cells and in vivo Effects after Intravenous Administration

Abstract

Gelatinase B is a matrix metalloproteinase involved in tissue remodelling. When mouse cells are triggered in vitro with interleukin-I, bacterial endotoxin, virus-mimicking double-stranded RNA or cytokine inducers, they produce gelatinase B. To test the effects of gelatinase B in vivo, the enzyme was expressed in Chinese hamster ovary (CHO) cells. Hybrid genomic DNA-cDNA constructs under the control of two constitutive viral promoters were generated by PCR-mediated exon amplification. In vitro transcription and translation of the mRNA in reticulocyte lysate yielded the correct 79-kDa protein, and expression in CHO cells resulted in an intact glycosylated 110-kDa gelatinase B which was enzymically active. However, the production yields of recombinant enzyme from 50 tested clones were low and cell-culture supernatants contained significant amounts of copurifiable endogenous CHO gelatinase B. Therefore, the enzyme was expressed in the yeast Pichia pastoris. Recombinant proenzyme was secreted and recovered from the yeast culture medium at 10 mg/l. Amino-terminal sequence analysis indicated that affinity purification of the recombinant protein on gelatin-Sepharose yielded the expected N-glycosylated proenzyme form (1 10 kDa) in addition to an amino-terminally truncated unglycosylated variant (69 kDa). Both forms had gelatinolytic activity on zymography. The recombinant mouse gelatinase B was used to determine its pharmacokinetics and its haematological effects in vivo. After intravenous injection in rabbits, gelatinase B disappeared from the circulation within 6 h. In addition to a transient leukopenia, we observed a rapid increase in leukocytosis, which indicates that gelatinase B might be a factor involved in the desorption of adherent leukocytes from the vascular bed and in the release of leukocytes from the bone marrow. Gelatinase B secretion and activation might well be one of the crucial molecular mechanisms explaining leukocytosis which is associated with infections and almost all types of inflammation.Keywords: matrix metalloproteinase; gelatinase B ; Pichia pastoris; expression ; leukocytosis.The matrix metalloproteinase (MMP) family is an expanding group of extracellular matrix-degrading enzymes, including secreted proteases (collagenases, gelatinases, stromelysins) and recently characterized membrane-type metalloproteinases (MT-MMP;Woessner, 1991 ;Murphy and Docherty, 1992; Sato and Seiki, 1996). The members of this enzyme family are capable of degrading most extracellular matrix components and are thus important mediators in physiological and pathological tissue remodelling (including tumor cell invasion and metastasis and autoimmune diseases) Van Damme, 1992, 1994;Docherty et al., 1992;MacDougall and Matrisian, 1995). Tight regulation of matrix degradation is essential and is achieved pretranslationally and posttranslationally at different levels. This regulation includes transcriptional control by cytokines and other mediators, specific extracellular or membrane-bound activation of proenzyme forms by a...

Expression of Matrix Metalloproteinases Subsequent to UrogenitalChlamydia muridarumInfection of Mice

“…3A). We ascribed this activity to MMP-9 due to its proximity to human MMP-9; its apparent molecular mass in zymograms corresponded to murine MMP-9, which has been estimated at 105 to 110 kDa (25,41), and the presence of a comigrating MMP-9-specific signal on a mouse-specific MMP-9 Western blot (see Fig. 4, below).…”

Migration across the sinusoidal endothelium regulates neutrophil mobilization in response to ELR + CXC chemokines

“…The Authors opoietic progenitor cells (Pruijt et al, 1999;McQuibban et al, 2001;Heissig et al, 2002), and granulocytes (Kjeldsen et al, 1992;Masure et al, 1997) from the bone marrow. Indeed, it has been proposed that an important step in leucocyte exit from the bone marrow is the degradation of the extracellular matrix within the bone marrow stroma (Opdenakker et al, 1998).…”

“…These data are apparently at odds with a previous study reporting that i.v. injection of recombinant MMP-9 elicits a blood leucocytosis (Masure et al, 1997). However, it is possible that the leucocytosis generated by the i.v.…”

“…Thus, it has been shown that an intravenous injection of recombinant neutrophil gelatinase B (MMP-9) induces a leucocytosis in vivo (Masure et al, 1997), whilst a clinical study reported a correlation between serum levels of human MMP-9 and circulating levels of neutrophils (Kjeldsen et al, 1992). The present study used a broad spectrum MMP-9 inhibitor (BB94) to investigate the role of MMPs in neutrophil mobilization stimulated by ELR + CXC chemokines.…”

Neutrophil-derived MMP-9 mediates synergistic mobilization of hematopoietic stem and progenitor cells by the combination of G-CSF and the chemokines GROβ/CXCL2 and GROβT /CXCL2Δ4

“…and S.F., unpublished observations, January 2003). Furthermore, administration of recombinant MMP-9 to rabbits induces a rapid peripheral leukocytosis, 67 although HSCs were not quantitated. It is likely that in GRO␤/GRO␤ T -mobilized mice, elevation of marrow MMP-9 that degrades matrix and releases HSCs, plus disruption of endothelial junctions by plasma MMP-9, coordinately result in HSC transmigration.…”