Systemic lupus erythematosus: molecular cloning of several recombinant DNase monoclonal kappa light chains with different catalytic properties

Abstract

An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of three patients with systemic lupus erythematosus was used. Phage particles displaying DNA binding light chains were isolated by affinity chromatography on DNA-cellulose, and the fraction eluted by an acidic buffer (pH 2.6) was used for preparation of individual monoclonal light chains (MLChs, 28 kDa). Thirty three of 687 individual colonies obtained were randomly chosen for study of MLCh DNase activity. Nineteen of 33 clones contained MLChs with DNase activity. Four preparations of MLChs were expressed in Escherichia coli in soluble form, purified by metal chelating chromatography followed by gel filtration, and studied in detail. Detection of DNase activity after SDS-PAGE in a gel containing DNA demonstrated that the four MLChs are not contaminated by canonical DNases. The MLChs demonstrated one or two pH optima. They were inactive after the dialysis against ethylenediaminetetraacetic acid but could be activated by several externally added metal ions; the ratio of relative activity in the presence of Mg(2+) , Mn(2+) , Ni(2+) , Ca(2+) , Zn(2+) , and Co(2+) was individual for each MLCh preparation. K(+) and Na(+) inhibited the DNase activity of various MLChs at different concentrations. Hydrolysis of DNA by all four MLCh was saturable and consistent with Michaelis-Menten kinetics. These clones are the first examples of recombinant MLChs possessing high affinity for DNA (Km = 3-9 nM) and demonstrating high kcat values (3.4-6.9 min(-1) ). These observations suggest that the systemic lupus erythematosus light chain repertoire can serve as a source of new types of DNases.

Catalytic Antibodies in Norm and Systemic Lupus Erythematosus

“…Na + and K + suppressed DNA-hydrolyzing activity of these MLChs at different concentrations [112]. Hydrolysis of DNA by all MLChs was consistent with Michaelis-Menten kinetics.…”

“…In this case, 33 of 687 individual colonies were chosen randomly for study of MLChs. Nineteen of 33 clones (58%) demonstrated DNase activity [112]. Detection of DNase activity in situ after SDS-PAGE of purified MLChs using gel containing DNA showed that they are not contaminated by canonical DNases.…”

“…Hydrolysis of DNA by all MLChs was consistent with Michaelis-Menten kinetics. These recombinant MLChs demonstrated high affinity for DNA (K m = 3-9 nM) and high k cat values (3.4-6.9 min −1 ) [112]. Even if we assume that each of the above-mentioned 16 fractions can contain only about 20 MLChs with DNase activity, their total number may be close to 300, but it is obvious that this number is much larger and can be close from one to several thousands.…”

“…For amplification of the phage library Escherichia coli TG1 presenting monoclonal light chains (MLChs) on the surface of phage particles was used [111,112]. Phage particles containing a pool of various MLChs having different affinity for DNA were separated by chromatography on DNA-cellulose ( Figure 10A).…”

“…Taking into account the fact that only 45 of 451 colonies were analyzed, it should be assumed that even this fraction contains much greater number of monoclonal abzymes with DNase activity. In this regard it should be mentioned the data of other article [112]. After separation of phage particles on DNA-cellulose, the fraction eluted by an acidic buffer (pH 2.6) was used for obtaining of MLChs (~28 kDa) with DNase activity.…”