Multiple sites of the cleavage of 21- and 25-mer encephalytogenic oligopeptides corresponding to human myelin basic protein (MBP) by specific anti-MBP antibodies from patients with systemic lupus erythematosus

Abstract

IgGs from patients with multiple sclerosis and systemic lupus erythematosus (SLE) purified on MBP-Sepharose in contrast to canonical proteases hydrolyze effectively only myelin basic protein (MBP), but not many other tested proteins. Here we have shown for the first time that anti-MBP SLE IgGs hydrolyze nonspecific tri- and tetrapeptides with an extreme low efficiency and cannot effectively hydrolyze longer 20-mer nonspecific oligopeptides corresponding to antigenic determinants (AGDs) of HIV-1 integrase. At the same time, anti-MBP SLE IgGs efficiently hydrolyze oligopeptides corresponding to AGDs of MBP. All sites of IgG-mediated proteolysis of 21-and 25-mer encephalytogenic oligopeptides corresponding to two known AGDs of MBP were found by a combination of reverse-phase chromatography, TLC, and MALDI spectrometry. Several clustered major, moderate, and minor sites of cleavage were revealed in the case of 21- and 25-mer oligopeptides. The active sites of anti-MBP abzymes are localised on their light chains, while heavy chains are responsible for the affinity of protein substrates. Interactions of intact globular proteins with both light and heavy chains of abzymes provide high affinity to MBP and specificity of this protein hydrolysis. The affinity of anti-MBP abzymes for intact MBP is approximately 1000-fold higher than for the oligopeptides. The data suggest that all oligopeptides interact mainly with the light chains of different monoclonal abzymes of total pool of IgGs, which possesses a lower affinity for substrates, and therefore, depending on the oligopeptide sequences, their hydrolysis may be less specific than globular protein and can occur in several sites.

Figure 1. TLC analysis of the hydrolysis of different OPs.

A, Boc-Val-Leu-Lys-MCA (shOP1), Pro-Phe-Arg-MCA (shOP2), and Boc-Ile-Glu-Gly-Arg-MCA (shOP3) (5 mM) were incubated for 24 h without Abs (lanesC) and in the presence of 0.05 mg/ml MS IgG_mix or SLE IgG_mix preparations (lanes shown on the panel) demonstrating comparable relative activities in the hydrolysis of intact MBP. B, Nonspecific in-OP1 and in-OP2 were incubated for 24 h without Abs (lanes 1) and in the presence of 0.05 mg/ml SLE IgG_mix (lanes 2); lanes C correspond to in-OP1 and in-OP2 before incubation. C, Specific X-OP21 (C) and X-OP25 oligopeptides were incubated for 7 h with hd-IgG_mix (lane C) and in the presence of 0.02 mg/ml sle-IgG_mix. These OPs were used in different concentrations (mM): 0.05 (lanes 1), 0.1 (lanes 2), 0.2 (lanes 3), 0.3 (lanes 4), 0.4 (lanes 5), and 0.5 (lanes 6).

Figure 2. Determination of the K_m and V _max values for OP21 (A) and OP25 (B) in the reaction catalyzed by sle-IgG_mix (0.1 µM) using a Lineweaver–Burk plot.

The reactions were performed as described in Materials and Methods. The average error in the initial rate determination from two experiments for each substrate concentration did not exceed 7–10%.

Figure 3. Profile of RPhC of SLE IgG_mix-dependent products of X-OP21 relatively deep hydrolysis (A) and analysis of X-OP products of the hydrolysis corresponding to different peaks after RPhC by TLC (B) or massspectrometry (C–E): (–), relative fluorescence (A).

Numbers of lines in panel B coincide with numbers of peaks on panel A; lanes C1, C2, and C3 correspond to the products of reaction mixture before their separation by RPhC, X-OP25 incubated in the absence of IgGs, and a free fluorescent compound X, respectively. The arrows (and numerals on panel B) indicate the positions of OPs of different length and compound X. Panels C, D, and E demonstrate the MALDI spectrum signals corresponding to the products eluted under RPhC in peaks 2, 5 and 6, respectively. See Materials and Methods for other details.

Figure 4. Profile of RPhC of sle-IgG_mix-dependent products of a complete hydrolysis of X-OP21 (A) and analysis of the products corresponding to different peaks after RPhC by TLC (B) or massspectrometry (C–E): (–), relative fluorescence (A).

Numbers of lines in panel B coincide with numbers of peaks on panel A; lane C1 corresponds to the products of reaction mixture before their separation by RPhC. The arrows (and numerals on panel B) indicate the positions of OPs of different length and compound X. Panels C, D, and E demonstrate the MALDI spectra corresponding to the products of the hydrolysis eluted under RPhC in peaks 2, 3, and 4, respectively (Fig. 4A). See Materials and Methods for other details.

Figure 5. Profile of RPhC of sle-IgG_mix-dependent products of X-OP25 relatively deep hydrolysis (A) and analysis of the products of the hydrolysis corresponding to different peaks after RPhC by TLC (B) or massspectrometry (C–E): (–), relative fluorescence (A).

Numbers of lines in panel B coincide with numbers of peaks on panel A; lanes C1, C2 and C3 correspond to the products of reaction mixture before their separation by RPhC, X-OP25 incubated in the absence of IgGs, and a free fluorescent compound X, respectively. The arrows (and numerals on panel B) indicate the positions of OPs of different length and compound X. Panel C demonstrates the MALDI spectrum of the products corresponding to peak 4, while panel D to peak 9 (Fig. 5A) and the intact X-OP25 before its hydrolysis, respectively. MALDI spectrum of all products of X-OP25 hydrolysis after 24 h of its incubation corresponding to non-fractionated reaction mixture is given on panel E. The length of the X-OPs is given on the bottom. See Materials and Methods for other details.

Figure 6. Complete sequence of human MBP (on the top) and all sites of cleavage of X-OP21 (C) and X-OP25 (F) determined using a combination of RPhC, TLC, and massspectrometry of detectable major and minor products of these OPs hydrolysis by sle-IgG_mix.

The positions of OP21 and OP25 sequences in the human MBP sequence are shown in bold. Panels A and D show all possible sites of these OPs cleavage by trypsin, while panels B and E demonstrate the major cleavage sites of MBP, which were found previously in the case of hydrolysis of globular intact MBP by MS IgGs . All sites corresponding to major and moderate products of the cleavage are shown by long and short arrows respectively, while to minor ones by diamonds (panels C and F).