Extrachromosomal genetic complementation of surface metalloproteinase (gp63)-deficient Leishmania increases their binding to macrophages.
A major surface glycoprotein of 63 kDa (gp63) has been previously identified biochemically and genetically as a zinc proteinase conserved in pathogenic Leishmania spp. The functional significance of this proteinase was analyzed by genetic approaches. A 15-kilobase DNA with a tunicamycin-resistance gene from Leishmania amazonensis was ligated in two different orientations into pBluescript containing a gp63 gene from Leishmania major. These plasmid constructs were used to transfect a variant of L. amazonensis deficient in gp63 expression. Both constructs were found to confer tunicamycin resistance with equal efficiency and remained structurally unchanged in the transfectants. RNA and immunoblot analyses showed over-expression of gp63 in the transfectants with one of the two plasmids constructed. The over-produced products were enzymatically active and expressed on the cell surface. Significantly, the transfectants with over-expressed gp63 increased by 2-fold over controls in their binding to macrophages. Evidence presented thus indicates that the gp63 gene constructed in the plasmid as described and introduced exogenously expresses in the gp63-deficient variants and that the expressed products are functionally active.
Liu
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X
Department of Microbiology/Immunology, University of Health Sciences/Chicago Medical School, IL 60064.
Chang
K P
KP
D000818:Animals; D002448:Cell Adhesion; D015870:Gene Expression; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D007891:Leishmania / Q000166:cytology* / Q000235:genetics; D008264:Macrophages / Q000469:parasitology*; D008666:Metalloendopeptidases / Q000502:physiology*; D010957:Plasmids; D015800:Protozoan Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection
D015800:Protozoan Proteins; D012333:RNA, Messenger; D008666:Metalloendopeptidases; C055146:glycoprotein gp63, Leishmania