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Comparative Study
. 2016 May 19;11(5):e0154688.
doi: 10.1371/journal.pone.0154688. eCollection 2016.

Comparison of Antibodies with Amylase Activity from Cerebrospinal Fluid and Serum of Patients with Multiple Sclerosis

Vasilii B Doronin  1 , Taisiya A Parkhomenko  2 , Massimiliano Castellazzi  3 , Edward Cesnik  3 , Valentina N Buneva  2   4 , Enrico Granieri  3 , Georgy A Nevinsky  2   4
Affiliations
Comparative Study

Comparison of Antibodies with Amylase Activity from Cerebrospinal Fluid and Serum of Patients with Multiple Sclerosis

Vasilii B Doronin et al. PLoS One. .

Abstract

We have recently shown that IgGs from serum and cerebrospinal fluid (CSF) of MS patients are active in hydrolysis of DNA and myelin basic protein. According to literature data, anti-DNA and anti-MBP abzymes may promote important neuropathologic mechanisms in this chronic inflammatory disorder and in MS pathogenesis development. At the same time, the involvement of antibodies with amylase activity in the pathogenesis of any autoimmune disease has not yet been identified. Electrophoretically and immunologically homogeneous IgGs were obtained by a sequential affinity chromatography of the CSF proteins on protein G-Sepharose and FPLC gel filtration. We are able to present the first unpredictable evidence showing that IgGs from CSF possess amylase activity and efficiently hydrolyze maltoheptaose; their average specific Ab activity is ~30-fold higher than that of antibodies from sera of the same MS patients. Specific average RA (SAA) for IgGs from healthy volunteers was approximately ~1000 lower than that for MS patients. In addition, it was shown that a relative SAA of total proteins of CSF (including Abs) ~15-fold lower than that for purified IgGs, while the relative SAA of the total sera protein is higher than that of sera IgGs by a factor of 1033. This result speaks in favor of the fact that amylolytic activity of CSF proteins is mainly caused by the activity of amylase abzymes. One cannot exclude, that amylase abzymes of CSF can play a, as yet unknown, role in the pathogenesis of MS. Some possible reasons of these findings are discussed.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1
SDS-PAGE analysis of homogeneity of csf-IgGmix (7 μg; lanes 2 and 3) corresponding to 15 CSFs of MS patients in 3–16% gradient gel before (lane 2) and after treatment with DTT (lane 4) followed by silver staining (A). Lanes 2 and 3 correspond to Western-blotting; Abs against human amylase were used in the case of IgG (lane 2) and human amylase (lane 3). The arrows (lane SP) indicate the positions of molecular mass markers. FPLC gel filtration of csf-IgGmix on a Superdex 200 column in an acidic buffer (pH 2.6) destroying immunocomplexes after Abs incubation in the same buffer (B): (—), absorbance at 280 nm (A280); (□), relative activity (RA) of IgGs in the hydrolysis of MHO. A complete hydrolysis of MHO was taken for 100%. In-gel assay of MHO-hydrolyzing activity of csf-IgGmix (■; 15 μg) of MS patients. The relative MHO -hydrolyzing activity (RA, %) was revealed using the extracts of 2-3-mm fragments of one longitudinal slice of the gel. The RA of IgGs corresponding to complete hydrolysis of MHO was taken for 100%. The second control longitudinal slice of the same gel was stained with Coomassie Blue (lane 1). Lane C shows positions of protein markers. TLC analysis of the hydrolysis of MHO by IgGs from CSFs of different MS patients (D). MHO (1.67 mM) was incubated for 6 h at 37°C without Abs (lanes C2) and in the presence of 0.07 mg/ml IgGmix from the plasma of healthy donors (lane C1) as well as individual IgGs (0.025 mg/ml, 2 h) from CSFs of different MS patients (lanes 1–5). The average error in the initial rate determination from three experiments did not exceed 7–10%. For details, see Materials and methods.
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Grants and funding

This research was made possible by grants mainly from Russian Science Foundation (No 16-15-10103 to G. A. Nevinsky), from the Presidium of the Russian Academy of Sciences (Molecular and Cellular Biology Program, 6.2 to GAN); purification of IgGs was made using grants from Russian Foundation for Basic Research (No. 16-04-00603, and 16-04-00604) and collection of samples by funds from the Regione Emilia Romagna, Italy (Ricerca Sanitaria Finalizzata; to EG). The funders had no role in study design, data collection and analysis, decision to publish.