Absence of autoantibodies against correctly folded recombinant fibrillin-1 protein in systemic sclerosis patients

Jürgen Brinckmann,Dieter P Reinhardt,Nico Hunzelmann,Ehab El-Hallous,Sven Krengel,Lynn Y Sakai,Thomas Krieg

doi:10.1186/ar1813

Abstract

Autoantibodies against short recombinant fragments of fibrillin-1 produced in bacterial expression systems have been found in tight-skin mouse, systemic sclerosis, mixed connective tissue disease, and primary pulmonary hypertension syndrome. In patients with scleroderma, the frequency of anti-fibrillin-1 antibodies was 42% in Caucasians. Until now it has been unclear whether this immune response has a primary function in disease pathogenesis or is a secondary phenomenon. In the present study we analyzed the frequency of autoantibodies against two overlapping recombinant polypeptides spanning the N-terminal and C-terminal halves of human fibrillin-1, which were produced in human embryonic kidney (HEK-293) cells. Correct three-dimensional structures of the recombinant fibrillin-1 polypeptides were shown by electron microscopy and immunoreactivity with antibodies. Screening of fibrillin-1 antibodies was performed in 41 sera from systemic sclerosis patients and in 44 healthy controls with a Caucasian background. Microtiter plates were coated with the recombinant polypeptides of fibrillin-1 and incubated with 1:100 diluted sera. Positive binding was defined as being more than 2 SD above the mean of the control group. ELISAs showed that none of the sera of patients with systemic sclerosis contained autoantibodies against the N-terminal or C-terminal recombinant fibrillin-1 polypeptide. The data show the absence of autoantibodies against recombinant fibrillin-1 protein in Caucasian systemic sclerosis patients. Because the correct three-dimensional folding of the recombinant proteins has been substantiated by several independent methods, we conclude that autoantibodies against correctly folded fibrillin are not a primary phenomenon in the pathogenesis of systemic sclerosis.

Highlights

Systemic sclerosis (SSc) is a connective tissue disease characterized by an excess deposition of collagen in skin and/or internal organs leading to malfunction and organ failure
Ultrastructural analysis of recombinant fibrillin-1 polypeptides To analyze the molecular shape of the recombinant polypeptides, they were revealed by electron microscopy after rotary shadowing (Fig. 1)
These results showed thread-like extended molecules for the recombinant polypeptides rF16 and rF6H representing the N-terminal and C-terminal halves of Roshfeahcduoommwabinningfaibnrtilalinm-i1now-teerremainal y(zrFe1d6b)y(aaen)ldeacnctdarorcbnaomrxbyio-ctxreoyr-smtceionrmaplyin(arFflt6(erHrFr)6ohHta)lr(vybe)s halves of human fibrillin-1 were analyzed by electron microscopy after rotary shadowing

Summary

Introduction

Systemic sclerosis (SSc) is a connective tissue disease characterized by an excess deposition of collagen in skin and/or internal organs leading to malfunction and organ failure. One hallmark of the disease is the presence of circulating autoantibodies against non-organ-specific nuclear and nucleolar antigens, which can be detected in at least 95% of patients. They include anti-centromere, anti-topoisomerase I and anti-RNA polymerase antibodies and are associated with distinct disease subtypes [1]. Heterozygous tight-skin mice (Tsk/+) are characterized by a phenotype of skin thickening and visceral fibrosis due to an increased deposition of extracellular matrix proteins in skin and organs. In a similar manner to human SSc, Tsk/+ mice produce autoantibodies against SSc-spe-

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Conclusion