A proteomic repertoire of autoantigens identified from the classic autoantibody clinical test substrate HEp-2 cells

Michael H Roehrl,Michael W Roehrl,Julia Y Wang,Jung-Hyun Rho,Wei Zhang

doi:10.1186/s12014-020-09298-3

Michael H Roehrl, Michael W Roehrl + Show 3 more

Open Access

https://doi.org/10.1186/s12014-020-09298-3

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Abstract

BackgroundAutoantibodies are a hallmark of autoimmune diseases. Autoantibody screening by indirect immunofluorescence staining of HEp-2 cells with patient sera is a current standard in clinical practice. Differential diagnosis of autoimmune disorders is based on commonly recognizable nuclear and cytoplasmic staining patterns. In this study, we attempted to identify as many autoantigens as possible from HEp-2 cells using a unique proteomic DS-affinity enrichment strategy.MethodsHEp-2 cells were cultured and lysed. Total proteins were extracted from cell lysate and fractionated with DS-Sepharose resins. Proteins were eluted with salt gradients, and fractions with low to high affinity were collected and sequenced by mass spectrometry. Literature text mining was conducted to verify the autoantigenicity of each protein. Protein interaction network and pathway analyses were performed on all identified proteins.ResultsThis study identified 107 proteins from fractions with low to high DS-affinity. Of these, 78 are verified autoantigens with previous reports as targets of autoantibodies, whereas 29 might be potential autoantigens yet to be verified. Among the 107 proteins, 82 can be located to nucleus and 15 to the mitotic cell cycle, which may correspond to the dominance of nuclear and mitotic staining patterns in HEp-2 test. There are 55 vesicle-associated proteins and 12 ribonucleoprotein granule proteins, which may contribute to the diverse speckled patterns in HEp-2 stains. There are also 32 proteins related to the cytoskeleton. Protein network analysis indicates that these proteins have significantly more interactions among themselves than would be expected of a random set, with the top 3 networks being mRNA metabolic process regulation, apoptosis, and DNA conformation change.ConclusionsThis study provides a proteomic repertoire of confirmed and potential autoantigens for future studies, and the findings are consistent with a mechanism for autoantigenicity: how self-molecules may form molecular complexes with DS to elicit autoimmunity. Our data contribute to the molecular etiology of autoimmunity and may deepen our understanding of autoimmune diseases.

Highlights

Autoantibodies are a hallmark of autoimmune diseases
We have proposed a uniform autoantigenicity mechanism by which autoAgs share a common biochemical property in their affinity to dermatan sulfate (DS), and by which autoAgs forming a molecular complex with DS to induce autoreactive B cell responses [1,2,3,4,5]
DS affinity strongly enriches for autoantigenic proteins Overall, this study identified a total of 107 proteins from DS-affinity enrichment of HEp-2 cellular extracts (Table 1)

Summary

Introduction

Autoantibodies are a hallmark of autoimmune diseases. Autoantibody screening by indirect immuno‐ fluorescence staining of HEp-2 cells with patient sera is a current standard in clinical practice. We attempted to identify as many autoantigens as possible from HEp-2 cells using a unique proteomic DS-affinity enrichment strategy. Autoimmune diseases occur when an immune system starts to attack its own body. The immune system reserves immune responses to attack invading microorganisms and protect the body. The immune response can sometimes go astray and mistakenly attack its own tissue. From a molecular point of view, certain self-molecules are targeted, as if they are non-self, by autoreactive cells, autoantibodies (autoAbs), or other factors, which subsequently leads to damage of the tissue where the autoantigens (autoAgs) reside

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