Intrapersonal and Populational Heterogeneity of the Chemokine RANTES

Abstract

BACKGROUND Current immunoassays for the chemokine RANTES (regulated on activation, normal T-cell expressed and secreted) are not tailored for specific isoforms that exist endogenously, despite the fact that variants with modified activity are known to exist. This is surprising in view of this protein’s ubiquitous increased presence in many diseases and that the 2 established isoforms are truncated by enzymes also correlated to disease. An in-depth population survey of RANTES heterogeneity in the context of multiple diseases via a mass spectrometric immunoassay (MSIA) may resolve this issue. METHODS We developed an MSIA for RANTES and endogenous variants apparent in human plasma. Samples from multiple cohorts of individuals (type 2 diabetes, congestive heart failure, history of myocardial infarction, and cancer patients) were run in parallel with samples from healthy individuals (239 people total). We used 230 μL of plasma per individual and tabulated relative percent abundance (RPA) values for identified isoforms. RESULTS We detected at least 19 variants, including the dipeptidyl peptidase IV (DPP-IV)-truncated variant. The majority of variants were unreported in the literature. Identifiable modifications included N- and/or C-terminal truncations, oxidation, glycation, and glycosylation. We observed statistically significant differences in RPA values for multiple variants between disease cohorts and recognized prospective disease-specific protein profiles for RANTES. CONCLUSIONS Because of widespread interest in the clinical value of RANTES, the protein diversity established here may aid in the design of future, fully quantitative assays. Equally important, an inclusive qualitative understanding of RANTES heterogeneity may present new insights into the relationship between RANTES and disease.

Calpain inhibition stabilizes the platelet proteome and reactivity in diabetes

“…More than 19 variants of CCL5 are known to exist, 32 and proteases such as dipeptidyl peptidase IV and cathepsin G can generate 3-68 and 4-68 variants of CCL5. 33,34 Although the clinical significance of these variants is unclear, higher plasma levels of CCL5 (3-68) have been reported in patients with type 2 diabetes.…”

“…33,34 Although the clinical significance of these variants is unclear, higher plasma levels of CCL5 (3-68) have been reported in patients with type 2 diabetes. 32 However, the latter peptide was reported to show reduced rather than enhanced activity toward cells expressing the recombinant CCR5 receptor. 33 This was not unexpected, because the N-terminus of many chemokines plays a critical role in the recognition and functional activation of cognate receptors.…”

Quantitative mass spectrometric immunoassay for the chemokine RANTES and its variants

“…RANTES is released from activated macrophages and T-lymphocytes, endothelial and epithelia cells, dermal fibroblast and renal tubular epithelium [10–12]. RANTES functional role has been demonstrated in association with autoimmune diseases [13], arthritis [14], diabetes [15], obesity and metabolic syndrome [16] and breast and cervical cancer [17]. In recent years RANTES and its proteoforms have been associated with cardiovascular diseases, including unstable carotid plaque [18] acute coronary syndrome [19] and atherosclerosis [20, 21].…”

“…RANTES bioactivity has historically been associated with the full-length protein form, which is composed of 68 amino acids, has two intact disulfide bonds and a molecular mass of 7,847 Da [14]. The biological activity is modulated by at least two pathways of posttranslational proteolysis [26].…”

“…When optimized, MSIA can provide detailed insights into the intrinsic protein characteristics, and has thus far been incorporated in numerous qualitative and a handful of fully quantitative assays [46–50]. We have previously developed a qualitative RANTES MSIA assay for high-throughput analysis of RANTES and its proteoforms [14]. The assay was used in screening a small population (~ 240 patients; healthy and diseased), in which the presence of previously identified variants was confirmed, and several new variants were discovered.…”