Comparison of Protein N-Homocysteinylation in Rat Plasma under Elevated Homocysteine Using a Specific Chemical Labeling Method

• Published in: Molecules (Basel, Switzerland) Switzerland), 2016-09, Vol.21 (9), p.1195
• Main Authors: Zang, Tianzhu, Pottenplackel, Ligi Paul, Handy, Diane E, Loscalzo, Joseph, Dai, Shujia, Deth, Richard C, Zhou, Zhaohui Sunny, Ma, Jisheng
• Format: Article
• Language: English
• Subjects: Animals; biotin-aldehyde; Blood Proteins - metabolism; cardiovascular disease; Homocysteine - blood; hyperhomocysteinemia; Hyperhomocysteinemia - blood; neuropsychiatric disease; plasma; Plasma - metabolism; protein N-homocysteinylation; Protein Processing, Post-Translational; Rats; Vitamin B 12 Deficiency - blood; Western blotting
• ISSN: 1420-3049; 1420-3049
• DOI: 10.3390/molecules21091195

Elevated blood concentrations of homocysteine have been well established as a risk factor for cardiovascular diseases and neuropsychiatric diseases, yet the etiologic relationship of homocysteine to these disorders remains poorly understood. Protein N-homocysteinylation has been hypothesized as a contributing factor; however, it has not been examined globally owing to the lack of suitable detection methods. We recently developed a selective chemical method to label N-homocysteinylated proteins with a biotin-aldehyde tag followed by Western blotting analysis, which was further optimized in this study. We then investigated the variation of protein N-homocysteinylation in plasma from rats on a vitamin B12 deficient diet. Elevated "total homocysteine" concentrations were determined in rats with a vitamin B12 deficient diet. Correspondingly, overall levels of plasma protein N-homocysteinylation displayed an increased trend, and furthermore, more pronounced and statistically significant changes (e.g., 1.8-fold, p-value: 0.03) were observed for some individual protein bands. Our results suggest that, as expected, a general metabolic correlation exists between "total homocysteine" and N-homocysteinylation, although other factors are involved in homocysteine/homocysteine thiolactone metabolism, such as the transsulfuration of homocysteine by cystathionine β-synthase or the hydrolysis of homocysteine thiolactone by paraoxonase 1 (PON1), may play more significant or direct roles in determining the level of N-homocysteinylation.