PRODUCTION AND CHARACTERIZATION OF RECOMBINANT ACTIVE-MOUSE GELATINASE-B FROM EUKARYOTIC CELLS AND IN-VIVO EFFECTS AFTER INTRAVENOUS ADMINISTRATION

Gelatinase B is a matrix metalloproteinase involved in tissue remodell ing. When mouse cells are triggered in vitro with interleukin-1, bacte rial endotoxin, virus-mimicking double-stranded RNA or cytokine induce rs, they produce gelatinase B. To test the effects of gelatinase B in vivo, the enzyme was expressed in Chinese hamster ovary (CHO) cells. H ybrid genomic DNA-cDNA constructs under the control of two constitutiv e viral promoters were generated by PCR-mediated exon amplification. I n vitro transcription and translation of the mRNA in reticulocyte lysa te yielded the correct 79-kDa protein, and expression in CHO cells res ulted in an intact glycosylated 110-kDa gelatinase B which was enzymic ally active. However, the production yields of recombinant enzyme from 50 tested clones were low and cell-culture supernatants contained sig nificant amounts of copurifiable endogenous CHO gelatinase B. Therefor e, 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 puri fication of the recombinant protein on gelatin-Sepharose yielded the e xpected N-glycosylated proenzyme form (110 kDa) in addition to an amin o-terminally truncated unglycosylated variant (69 kDa). Both forms had gelatinolytic activity on zymography. The recombinant mouse gelatinas e 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 transien t leukopenia, we observed a rapid increase in leukocytosis, which indi cates that gelatinase B might be a factor involved in the desorption o f adherent leukocytes from the Vascular bed and in the release of leuk ocytes from the bone marrow. Gelatinase B secretion and activation mig ht well be one of the crucial molecular mechanisms explaining leukocyt osis which is associated with infections and almost all types of infla mmation.