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Published ahead of print on December 23, 2003, doi:10.1165/rcmb.2003-0139OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 801-807, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0139OC

Design and Use of Highly Specific Substrates of Neutrophil Elastase and Proteinase 3

Brice Korkmaz, Sylvie Attucci, Thierry Moreau, Emmanuel Godat, Luiz Juliano and Francis Gauthier

INSERM U618, Protéases et Vectorisation Pulmonaires, University François Rabelais, Tours, France; and Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil

Address correspondence to: Francis Gauthier, INSERM U618 "Proteases et Vectorisation Pulmonaires," University François Rabelais, 2 bis Bd Tonnellé, 37032 Tours Cedex, France. E-mail: gauthier{at}univ-tours.fr

We have exploited differences in the structures of S2' subsites of proteinase 3 (Pr3) and human neutrophil elastase (HNE) to prepare new fluorogenic substrates specific for each of these proteases. The positively charged residue at position 143 in Pr3 prevents it from accommodating an arginyl residue at S2' and improves the binding of P2' aspartyl-containing substrates, as judged by the decreased Km. As a result, the kcat/Km for Abz-VADCADQ-EDDnp is over 500 times greater for Pr3 than for HNE, and that for Abz-APEEIMRRQ-EDDnp is over 500 times greater for HNE than for Pr3. This allows each protease activity to be measured in the presence of a large excess of the other, as might occur in vivo. Placing a prolyl residue in position P2' greatly impaired substrate binding to both HNE and Pr3, which further emphasizes the importance of S' subsites in these proteases. HNE and Pr3 activities were measured with these substrates at the surface of fixed polymorphonuclear leukocytes (PMNs) before and after activation. This demonstrated that their active site remains accessible when they are exposed to the cell surface. Both membrane-bound proteases were strongly inhibited by low Mr serine protease inhibitors, but only partially by inhibitors of larger Mr such as {alpha}1-protease inhibitor, the main physiologic inhibitor in lung secretions. Most of membrane-bound HNE and Pr3 can be released from the membrane surface of fixed cells by a buffer containing detergent, suggesting that hydrophobic interactions are involved in membrane binding.

Abbreviations: {alpha}1-protease inhibitor, {alpha}1-PI • ortho–aminobenzoic acid, Abz • 7-amino-4-trifluoromethyl-coumarin, AFC • p-aminophenylmercuric acetate, APMA • bronchoalveolar lavage, BAL • chloromethyl ketone, CMK • cytokine-response modifier A, CrmA • N-(2,4-dinitrophenyl) ethylenediamine, EDDnp • L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-guanido)butane, E64 • fluorescein isothiocyanate, FITC • 9-fluoroenylmethoxycarbonyl, FMOC • human neutrophil elastase, HNE • high-performance liquid chromatography, HPLC • methoxysuccinyl, MeO-Suc- • turkey ovomucoid inhibitor domain 3, OMT3 • plasminogen activator inhibitor 1, PAI-1 • phosphate-buffered saline, PBS • polymorphonuclear leukocyte, PMN • phenylmethylsulfonyl fluoride, PMSF • proteinase 3, Pr3 • nitrotyrosine, Y(NO2)




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