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. 2006 Jan 10;103(2):281-6.
doi: 10.1073/pnas.0509849103. Epub 2005 Dec 30.

Autoantibodies to myelin basic protein catalyze site-specific degradation of their antigen

Natalia A Ponomarenko  1 , Oxana M Durova, Ivan I Vorobiev, Alexey A Belogurov Jr, Inna N Kurkova, Alexander G Petrenko, Georgy B Telegin, Sergey V Suchkov, Sergey L Kiselev, Maria A Lagarkova, Vadim M Govorun, Marina V Serebryakova, Bérangère Avalle, Pete Tornatore, Alexander Karavanov, Herbert C Morse 3rd, Daniel Thomas, Alain Friboulet, Alexander G Gabibov
Affiliations

Autoantibodies to myelin basic protein catalyze site-specific degradation of their antigen

Natalia A Ponomarenko et al. Proc Natl Acad Sci U S A. .

Abstract

Autoantibody-mediated tissue destruction is among the main features of organ-specific autoimmunity. This report describes "an antibody enzyme" (abzyme) contribution to the site-specific degradation of a neural antigen. We detected proteolytic activity toward myelin basic protein (MBP) in the fraction of antibodies purified from the sera of humans with multiple sclerosis (MS) and mice with induced experimental allergic encephalomyelitis. Chromatography and zymography data demonstrated that the proteolytic activity of this preparation was exclusively associated with the antibodies. No activity was found in the IgG fraction of healthy donors. The human and murine abzymes efficiently cleaved MBP but not other protein substrates tested. The sites of MBP cleavage determined by mass spectrometry were localized within immunodominant regions of MBP. The abzymes could also cleave recombinant substrates containing encephalytogenic MBP(85-101) peptide. An established MS therapeutic Copaxone appeared to be a specific abzyme inhibitor. Thus, the discovered epitope-specific antibody-mediated degradation of MBP suggests a mechanistic explanation of the slow development of neurodegeneration associated with MS.

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Figures

Fig. 1.
Fig. 1.
IgGs of MS patient and EAE mice react with MBP in rat brain sections. (A) Western blotting of purified MBP (m) and rat brain tissue homogenate (h) with antigen affinity-purified IgG from a MS patient, a EAE SJL mouse, and with MAB382. (B) Schematic myelin distribution in a rat brain section. (C) Wright's stain of the rat brain section chosen for analysis. D-H were taken from the indicated areas. (D-F) Double-label immunofluorescence of mouse anti-MBP monoclonal antibody MAB382 and catalytic IgG from MS patient. (D) MAB382 (Alexa 546, red). (E) MS (FITC, green). (F) Merge of D and E.(G and H) Immunofluorescence of MAB382 and IgG from EAE SJL mouse on serial sections. (G) MAB382 (Alexa 546, red). (H) IgG from EAE mouse (Alexa 488, green).
Fig. 2.
Fig. 2.
MBP degradation by purified autoantibodies. MBP affinity-purified antibodies from sera of the MS patient and EAE SJL and EAE C57BL/6 mice were incubated with purified MBP (A) and MOG (B) followed by SDS/PAGE analysis and Coomassie staining. IgG mbp+, MBP-binding antibodies. IgG heavy chains, light chains, and noncleaved MBP bands are marked as Hc, Lc, and MBP, respectively. (C) The MBP autoantibodies were separated by 5-20% gradient SDS/PAGE with FITC-BSA fluorescent substrate impregnated in the separating gel. After electrophoresis, proteins were in-gel renatured by Triton X-100 washes. Proteolytic degradation was visualized by fluorescence intensity increase, seen as bright bands on the dark background. Bovine trypsin (10 and 30 pg) was used as a positive control. IgG mbp+, MBP-binding antibodies; mbp-, antibodies not bound to the MBP-affinity column. (D) MBP degradation by the purified Fab fragment derived from the whole IgG of the MS patient visualized by SDS/PAGE.
Fig. 3.
Fig. 3.
Analysis of major MBP cleavage products. Reverse-phase HPLC-MS analysis of major MBP cleavage products. Column eluate fractions, corresponding to dominant chromatography peaks (A), were collected, freeze dried, redissolved, and applied to SELDI H4 chip and tricine-SDS/PAGE (B). Gel was stained by Sypro Orange. Peptides, unambiguously identified by SELDI and clearly seen in corresponding gel lanes, are indicated. (C) Schematic description of the preferential antibody cleavage sites in the MBP sequence. Sequence fragments identical to the immunodominant MBP-derived peptides (12-31, 82-98, 110-128, and 144-169) are shown in yellow rectangles, and the encephalitogenic peptide region (86-98) is marked by the red box. (D) 32P-MBP degradation by autoantibodies. Autoradiography of 32P-phosphorylated MBP hydrolysis by proteolytic mouse (EAE SJL) and human (MS) IgG. Line M, molecular mass markers (range 2.5-16.9 kDa, Amersham Pharmacia).
Fig. 4.
Fig. 4.
The specificity of the anti-MBP antibodies toward recombinant substrates. Schematic description of the designed fusion proteins (A) Identification of the cleavage sites of Trx-MBP fusion proteins and nonmodified Trx generated by anti-MBP antibodies: SJL mice with EAE (white boxes), MS patient (red boxes), and C57BL/6 mice with EAE (blue boxes) (B). Data obtained by mass-spectrometry analysis and gel electrophoresis of degradation products (Fig. 8, which is published as supporting information on the PNAS web site).
Fig. 5.
Fig. 5.
Proteolytic activity of the MS patient and EAE mice antibodies is inhibited by glatiramer acetate (Copaxone). Purified MBP was incubated with either trypsin or IgGs of human (MS) or mouse (EAE) and a variable concentration of glatiramer acetate (Copaxone). The proteolytic activity was calculated as the ratio of degraded and nondegraded MBP measured by the densitometry of Coomassie-stained gel.
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