Olgica Trenchevska (Presenter)
Arizona State University
Bio: I have completed my PhD studies at the Faculty of Natural Sciences and mathematics in Skopje, Macedonia. Majority of my research work focuses on application of new mwthodologies for analyses of proteins as clinical biomarkers. In the past four years I have been working in the Molecular Biomarkers Lab at the Biodesign Institute at Arizona State University, Tempe, AZ. My research includes developing and optimizing methods for protein quantification, analyzing the analytical characteristics of the developed assays, and their implementation in biological samples for screening potential biomarker candidates. Currently I am extanding my focus on the translational aspects of MS application – focusing on the relevance of proteoforms as clinical biomarkers in association with different physiological and pathological states.
Authorship: Olgica Trenchevska (1), Hussein Yassine (2), Peter D. Reaven (3), Juraj Koska (3), Randall W. Nelson (1) and Dobrin Nedelkov (1)
(1) Arizona State University, Tempe, AZ; (2) University of Southern California, Los Angeles, CA; (3) Phoenix Veteran Affairs Medical Center, Phoenix, AZ
Proteins can exist in multiple proteoforms in vivo in different physiological and pathological states. In this work, we addressed the potential of utilizing proteoforms as clinical markers, by exploring the association between apolipoprotein C-III (apoC-III) proteoforms and lipid metabolism. We used mass spectrometric immunoassay to perform cross-sectional and longitudinal characterization of apoC-III proteoforms in obese adolescents, and patients with impaired glucose tolerance and type 2 diabetes. We found strong inverse and independent associations between the relative amount of disialylated apoC-III and plasma triglycerides concentrations in all three cohorts. Other measures, such as cholesterol distribution, also correlated with apoC-III proteoforms.
Introduction: Protein biomarkers are widely utilized in clinical diagnosis, prognosis and treatment monitoring of numerous diseases. Most proteins are known to exist as multiple proteoforms in vivo as a result of posttranslational processing, polymorphism and/or genetic mutations. In 50% of proteins, posttranslational modifications (PTMs) have a major role in determining the protein characteristics and functions . Therefore, exploring proteoforms may be useful to determine their full potential and significance.
Apolipoprotein C-III (apoC-III) is a members of the C-apolipoprotein family, and is a main component of the very-low density lipoprotein (VLDL) fraction . ApoC-III inhibits the lipolysis of triglyceride (TG)-rich lipoproteins and is associated with elevated TG-rich lipoproteins . Recent studies indicate that loss-of-function mutations in the apoC-III gene are associated with reduced triglyceride levels and cardiovascular risk . ApoC-III is known to exist in multiple proteoforms in vivo, as a result of intensive glycosylation. There are a limited number of studies that exploit the role of apoC-III proteoforms; some are focused on the functional differences of apoC-III proteoforms in regulating liver VLDL uptake in vitro , while others analyze the distinct effects of major proteoforms on metabolic phenotypes .
Methods: Mass spectrometry-based methods can be used to study apoC-III proteoforms [7, 8], and correlate their structural modifications with function in physiological and pathological states. In our previous work, we have developed quantitative mass spectrometric immunoassay (MSIA) for analysis of apolipoprotein C-III, as part of a multiplexed assay together with apolipoproteins C-I and C-II . In this work we utilize this MSIA approach to perform a cross-sectional and longitudinal study of apoC-III proteoforms in plasma samples from obese adolescents (n=209), and adult patients with impaired glucose tolerance (n=531), and a cohort of patients with atherosclerosis and on intensive glycemic control (n=296) . Sufficient number of samples from a follow-up visit (n=253) were also available and analyzed for the patients on intensive glycemic control.
Results: A total of twelve apoC-III proteoforms were detected in all cohorts, including non-sialylated apoC-III0 sub forms (native - apoC-III0a and glycosylated - apoC-III0b); the most abundant proteoform – monosialylated apoC-III (apoC-III1), disialylated apoC-III (apoC-III2), as well as eight other apoC-III proteoforms that contained fucose in their sugar motif.
In all three cohorts we found strong inverse relationship between the relative abundance of the apoC-III2 proteoform (normalized to the apoC-III1) and the total triglyceride concentration. The association between apoC-III2 and triglycerides remained present after adjustment for relevant demographic factors and lipid lowering medications in the adult cohorts. In addition, higher apoC-III2 was also associated with an improved overall lipid profile, including lower liver fat in the obese adolescent patient cohort, and lower total and LDL cholesterol and higher HDL cholesterol in adult patient cohorts.
In the follow-up sample cohort, we examined the association between apoC-III2 with incident cardiovascular outcomes. Addition of apoC-III2 relative abundance to the Framingham score considerably improved the baseline clinical risk model, indicating a potential role for apoC-III2 in major adverse cardiac events (MACE) pathogenesis and/or prediction.
Conclusions: These analyses provide an evidence, from three independent cohorts, of a strong and inverse association between relative amounts of a disialylated apoC-III proteoform and proatherogenic plasma lipid profiles. Measuring plasma apo C-III proteoforms abundance may be used as an independent determinant of plasma lipid metabolism, as well as a predictor marker for cardiovascular events.
This research was funded in part by NIH grants R01DK082542 and R24DK090958.
References & Acknowledgements:
 Prabakaran, S., Lippens, G., Steen, H., Gunawardena, J., Post-translational modification: nature's escape from genetic imprisonment and the basis for dynamic information encoding. Wiley Interdiscip Rev Syst Biol Med 2012, 4, 565-583.
 Jong, M. C., Hofker, M. H., Havekes, L. M., Role of ApoCs in lipoprotein metabolism: functional differences between ApoC1, ApoC2, and ApoC3. Arterioscler Thromb Vasc Biol 1999, 19, 472-484.
 Zheng, C., Khoo, C., Furtado, J., Sacks, F. M., Apolipoprotein C-III and the metabolic basis for hypertriglyceridemia and the dense low-density lipoprotein phenotype. Circulation 2010, 121, 1722-1734.
 Crosby, J., Peloso, G. M., Auer, P. L., Crosslin, D. R., et al., Loss-of-function mutations in APOC3, triglycerides, and coronary disease. N Engl J Med 2014, 371, 22-31.
 Mann, C. J., Troussard, A. A., Yen, F. T., Hannouche, N., et al., Inhibitory effects of specific apolipoprotein C-III isoforms on the binding of triglyceride-rich lipoproteins to the lipolysis-stimulated receptor. J Biol Chem 1997, 272, 31348-31354.
 Hiukka, A., Ståhlman, M., Pettersson, C., Levin, M., et al., ApoCIII-enriched LDL in type 2 diabetes displays altered lipid composition, increased susceptibility for sphingomyelinase, and increased binding to biglycan. Diabetes 2009, 58, 2018-2026.
 Nicolardi, S., van der Burgt, Y. E., Dragan, I., Hensbergen, P. J., Deelder, A. M., Identification of new apolipoprotein-CIII glycoforms with ultrahigh resolution MALDI-FTICR mass spectrometry of human sera. J Proteome Res 2013, 12, 2260-2268.
 Wada, Y., Kadoya, M., Okamoto, N., Mass spectrometry of apolipoprotein C-III, a simple analytical method for mucin-type O-glycosylation and its application to an autosomal recessive cutis laxa type-2 (ARCL2) patient. Glycobiology 2012, 22, 1140-1144.
 Trenchevska, O., Schaab, M. R., Nelson, R. W., Nedelkov, D., Development of multiplex mass spectrometric immunoassay for detection and quantification of apolipoproteins C-I, C-II, C-III and their proteoforms. Methods 2015.
 Reaven, P. D., Moritz, T. E., Schwenke, D. C., Anderson, R. J., et al., Intensive glucose-lowering therapy reduces cardiovascular disease events in veterans affairs diabetes trial participants with lower calcified coronary atherosclerosis. Diabetes 2009, 58, 2642-2648.
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