Homocysteine-Thiolactone - Homocysteine in Protein Structure/Function and Human Disease - page 35

Table 3.5 Hcy-thiolactone levels in cultures of wild-type and temperature-sensitive aminoacyl-
tRNA synthetase mutants of Chinese hamster ovary (CHO) cells (data from [138])

Hcy-thiolactone, pmol/10 7 cells
34 C

39.5 C

CHO cell line

Wild type 2.0 3.7

1.6 16.4

Met-1 0.3

< 0.1

< 0.1
Arg-1 3.0 5.8
7.6 21.3
Hcy-thiolactone levels were measured at 34 C and 395 C after 6 h and 12 h of labeling with
10 μM[ 35 S]methionine (0.1 mCi mL 1 ,l0 4 Ci mol 1 ;1Ci¼ 37 GBq) in αMEM medium
supplemented with 5 % fetal bovine serum, 10

0.5

μgmL

1

μgmL

1

adenosine, and 10

deoxythymidine

evidence showing that enhanced Hcy-thiolactone biosynthesis is linked to patho-
logical conditions.

(a) Cells expressing MetRS variants defective in the Met-binding site of the
enzyme are also defective in Hcy-thiolactone synthesis as shown in Chinese
hamster ovary (CHO) cells [138], the yeast Saccharomyces cerevisiae [63], and
E. coli [197]. For example, Met-1 CHO cells expressing a temperature-sensitive
(ts) MetRS variant do not produce Hcy-thiolactone at the nonpermissive tem-
perature (39.5 C), while they support Hcy-thiolactone biosynthesis at the
permissive temperature (34 C), albeit to a lesser extent than wild-type cells
(Table 3.5 ) [138]. Control experiment with Arg-1 CHO cells expressing a ts
ArgRS variant shows that a mutation in unrelated aminoacyl-tRNA synthetase
does not prevent Hcy-thiolactone synthesis.
(b) Conversely, increasing the expression of MetRS in growing cells also leads to
proportional increases in Hcy-thiolactone synthesis as demonstrated for E. coli
[197], yeast [63], and rice [190] enzymes. A relationship between cellular
MetRS levels and the rate of Hcy-thiolactone synthesis in E. coli is illustrated
in Fig. 3.6 .
(c) Biosynthesis of Hcy-thiolactone in human endothelial cells is directly propor-
tional to Hcy concentration in culture media and inversely proportional to Met
concentration (Fig. 3.7 ), consistent with the involvement of MetRS. Supple-
mentation with folic acid decreases Hcy-thiolactone biosynthesis by lowering
Hcy and increasing Met concentrations in endothelial cells [74].
(d) Hyperhomocysteinemia caused by genetic or nutritional deficiencies in Hcy or
folate metabolism in all studied organisms, including human [93], mouse [93,
113], plant [190], yeast [63], and E. coli [197], leads to greatly increased
biosynthesis of Hcy-thiolactone. Comparisons of plasma Hcy-thiolactone
levels between cystathionine -synthase (CBS)-deficient or methylenetetrahy-

drofolate reductase (MTHFR)-deficient patients and unaffected control
individuals [93] are illustrated in Tables 3.6 and 3.12 .

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