Review
Novel combinations of Post-Translational Modification (PTM) neo-epitopes provide tissue-specific biochemical markers—are they the cause or the consequence of the disease?

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Abstract

The aim of this review is to discuss the potential usefulness of novel advances in the class of biochemical markers, neo-epitopes. Neo-epitopes are post-translational modifications (PTMs) of proteins formed by processes such as protease cleavage, citrullination, nitrosylation, glycosylation and isomerization. Each modification results from a specific local physiological or pathobiologial process. Identification of each modification, and the affected tissue-specific protein, may produce a unique disease-specific biochemical marker. One example of neo-epitopes detectable in protein fragments are type II collagen degradation products. These 2nd generation biochemical markers have proven useful for research on joint damage. Such neo-epitopes are being utilized in translational medicine to estimate safety and efficacy in both preclinical models and clinical settings. More advanced, 3rd generation biochemical markers, which may more accurately identify both the affected tissue and the disease stage, might be developed through research into multiple PTMs occurring during specific disease pathogenesis. The end-product of these investigations is one single measurement for each disease. To date, advanced biochemical markers have been identified in bone, cardiovascular, fibrosis and cartilage diseases and continue research in Alzheimer's and chronic obstructive pulmonary diseases. These advanced biochemical marker assays relying on analytes that are modified by multiple PTMs may become optimal tools that meet the BIPED (Burden of disease, Investigatory, Prognostic, Efficacy of intervention and Diagnosis) biochemical marker “usefulness” criteria. For some of these markers it may be interesting to also investigate whether the PTMs are the cause or consequence of a certain disease.

Introduction

Extracellular matrix (ECM) remodeling, in which old or damaged proteins are broken down and replaced by new, intact ones, is a key process in tissue homeostasis. Specific proteolytic activities are a prerequisite for a range of cellular functions and interactions within the extracellular matrix during this remodeling. These specific activities are tightly coordinated in physiological situations, in which a detailed sequence of events locates the adequate proteolytic response to promote tissue turnover. Under pathological situations, such as inflammation, fibrosis and cancer, this repair–response relationship is disturbed, leading to excessive tissue turnover and abnormal remodeling in which the original proteins of the extracellular matrix (ECM) are replaced by a different composition of ECM proteins. The quality of the matrix is altered as a consequence. This imbalanced ECM remodeling also leads to excessive levels of tissue- and pathology-specific turnover products released in the systemic circulation which, if detected, may be used as molecular biochemical markers of various pathologies.

During turnover of healthy tissue, specific proteases trigger specific sequences of cellular events within the ECM. For example, endopeptidases such as matrix metalloproteinases (MMPs) and cysteine proteases degrade ECM proteins including collagens and proteoglycans and in the process generate specific protein cleavage fragments. Even though many components of the ECM, as well as enzymes responsible for remodeling, are present in different tissues, the identification of specific proteases acting on specific ECM proteins may provide a combination that uniquely detects activity ongoing in a particular tissue or pathology. This increased accuracy results from a combination of pathological PTMs that may alter proteins in a pathologically specific manner, as will be carefully discussed.

Biochemical markers are receiving increasing attention for their potential to accurately and relatively quickly assess the efficacy of potential treatments and prognosis of patients diagnosed with chronic diseases such as osteoporosis, osteoarthritis and rheumatoid arthritis [58]. Biochemical markers may be divided into three generations according to the assay technologies used and approaches taken, as outlined in Table 1. First generation is based on total protein measurement often with polyclonal antibodies. Second generation is based on monoclonal antibodies directed against single, specific PTMs of different proteins. Third generation assays are sandwich assays including multiple PTMs, often based on different specific monoclonal antibodies. This separation of biochemical markers into different generations is important, as different technologies provide different levels of accuracy and precision. Furthermore, the recently identified PTM features (aging, citrullination, protease degradation, glycosylation and nitrosylation [21], [23] as will be carefully outlined) of some biochemical markers suggest that additional applied research is required to understand the exact analyte measured, as different PTMs may have different pathological meanings. The different PTMs may in part explain why different versions of analytical methods thought to be measuring the same protein often lead to divergent results. A well-characterized example of this issue is measurement of type I collagen and its degradation fragments in postmenopausal osteoporosis [71], [106]. Measurement of total type I collagen in serum would suggest that postmenopausal women have more type I collagen than age-matched individuals [71]. However, whether the increased levels are the consequence of degradation, or reduced formation of new type I collagen, is not revealed by a first-generation assay. Second generation assays separately assessing collagen formation and collagen degradation elegantly provide evidence that postmenopausal women have an increase in both type I collagen synthesis and degradation, but with a balance in favor of degradation, resulting in a continuous loss of bone [71]. This highlights the need for further understanding of the analyte measured, and its pathobiological basis.

Biochemical markers have been applied mainly in diseases that evolve slowly over many years. In osteoporosis and arthritis, bone resorption and cartilage degradation markers, the carboxy-terminal cross-linking telopeptide of type I collagen (CTX-I) and a carboxy-terminal cross-linking telopeptide of type II collagen (CTX-II), respectively, have been used extensively [58], [106], [127]. These biochemical markers have been categorized according to the recently proposed BIPED (Burden of disease, Investigative, Prognostic, Efficacy of intervention, and Diagnostic) classification, developed by the US National Institutes of Health (NIH)-industry partnership funded by the Osteoarthritis Biochemical markers Network [7]. The FDA critical path initiative launched in 2004 further emphasized the need for translational science and the use of biochemical markers during drug discovery and development [58].

The aim of this paper is to review and examine the use of existing biochemical markers and the need for new tools to more reliably describe tissue turnover in both healthy and disease-affected individuals. We conclude that third-generation biochemical markers derived from multiple PTMs of proteins can be combined to enhance the specificity of assessments of the states of both tissue and pathology. As a result of critically evaluating the nature of some of these PTMs, we also question whether modifications that may be tissue and disease specific may also be the cause of some aspects of pathogenesis or the consequence of the disease.

Section snippets

Post-translational modification—general considerations

Because PTMs are defined as modifications made subsequent to translation of the protein, most PTMs are not DNA-coded, but rather a consequence of tissue physiology and pathophysiology. This excludes some modifications that are protein-sequence specific, such as some glycosylations and phosphorylations [21], [23]. Protease-generated neo-epitopes have to date received more attention than other PTMs. However, potentially more advanced PTMs that are relatively specific for pathological conditions

Use of biochemical markers—sensitivity to change even in slowly progressing diseases

An example of a slowly progressing disease that may be difficult to diagnose in the early stages is OP, which is inherently linked to low bone mass. The level of bone mass is well integrated into determining the efficacy of new anti-osteoporotic drugs. However, in contrast to imaging techniques, biochemical markers of bone and cartilage turnover obtained in serum or urine samples, respectively, show changes in a markedly larger range compared with the imprecision of the assay (< 8–10%).

What are neo-epitopes?

We have briefly discussed the importance of biochemical markers and how these could be tissue and/or disease specific by identifying PTMs. PTMs are modifications to the composition or structure of proteins, and have been called neo-epitopes. Neo-epitopes are unique parts of a molecule that can be selected as a biochemical marker. Fig. 2 depicts a handful of different types of PTMs that have been identified in biochemical marker development. Pathologically relevant protein modifications are not

Biochemical marker classification—the BIPED

Previous paragraph suggests that optimal neo-epitope research projects may be designed for many different diseases by identification of the major pathologic proteases in combination with the predominant extracellular proteins in that tissue. Careful investigations for the expression and identification of different proteases and proteins, at different stages during progression of the disease, may produce biochemical markers that will not only comply with the BIPED criteria, but also enable

PTMs—the cause or consequence of the disease?

As highlighted in this focused review, proteins are complex molecules susceptible to numerous post-translational modifications occurring spontaneously or as a consequence of physiologic or pathologic processes. Today, it is well established that PTMs can present advanced epitopes and/or create neo-epitopes, to which the body have little or no tolerance for [21]. Antigenicity and interactions of proteins with components of the immune system may be profoundly affected by post-translational

Conclusion and perspectives

In this manuscript we have highlighted the possibilities that are emerging in clinical chemistry, by the combination of multiple disease-specific neo-epitopes in third-generation biochemical marker assays. This approach has already been applied to some disease areas such as bone and fibrosis, and may be advantageous in yet other disease areas. By incorporating the most optimal biochemical markers in all aspects of drug discovery and development, with the promise of translational science, novel

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