Human Erythrocyte Bisphosphoglycerate Mutase: Inactivation by Glycation In Vivo and In Vitro

Abstract

2,3-Bisphosphoglycerate mutase (BPGM) [EC 5.4.2.4] is a multifunctional enzyme that catalyzes both the synthesis and the degradation of 2,3-diphosphoglycerate (2,3-DPG) and contains three types of activities in that it functions as a 2,3-DPG synthetase, a phosphoglycerate mutase and a 2,3-DPG phosphatase. In humans, BPGM occurs only in erythrocytes and plays a pivotal role in the dissociation of oxygen from hemoglobin via 2,3-DPG. The present study shows that the specific activity of BPGM in erythrocytes of diabetic patients is decreased, compared to normal controls as judged by 2,3-DPG synthetase activity and immunoreactive contents. To understand the mechanism by which the enzyme is inactivated, the enzyme was purified from pooled erythrocytes from diabetic patients and subjected to a boronate affinity column. The flow through fraction was active while the bound fraction was completely inactive. The bound fraction was reactive to an anti-hexitollysine antibody, indicating that the enzyme had undergone glycation and inactivation. The primary glycated site of the enzyme was found to be Lys158 as judged by amino acid sequencing and the reactivity with an anti-hexitollysine IgG, after reverse-phase HPLC of the lysyl-endopeptidase-digested peptides. Extensive glycation of recombinant BPGM in vitro indicated that the glycation sites were Lys2, Lys4, Lys17, Lys42, Lys158, and Lys196. From these results, the loss of enzymatic activity appears to be due to the glycation of Lys158 which may be located in the vicinity of the substrate binding site.

Effect of prematurity on protein glycosylation in the newborn

“…Previous studies have reported that oxidative stress can increase the glycosylation of proteins 6,8,9 . We hypothesized that hemoglobin glycosylation would be higher in pre‐term infants for two reasons: first, because certain prematurity‐associated complications mimic the effects of increased glycosylation of proteins; 2–5 and second, pre‐term infants have reduced levels of antioxidants and elevated levels of oxidative stress compared with those of full‐term infants, and oxidative stress can increase glycosylation 10–14 . The present study showed, however, that GHb levels were lower in pre‐term than full‐term infants.…”

“…Glycosylation of proteins in general can modify protein structure and alter catalytic properties, thereby causing cellular dysfunction in many organs 2 . For example, glycosylation of proteins can contribute to the development of retinopathy; and glycosylation of hemoglobin can alter oxygen dissociation, which influences tissue oxygenation and thus development of the fetus 2–5 . Glycohemoglobins (GHb) are the products of irreversible non‐enzymatic reactions between glucose and the hemoglobin molecule 6,7 .…”

Individuality and variation in gene expression patterns in human blood

“…BCL2L1 (BCL-X), BNIP3L, and BAG1 are BCL2-associated proteins involved erythropoietin's known suppression of apoptosis during erythropoiesis (15). Other genes correlated with RDW are involved in the heme biosynthetic pathway (ALAS2) and in regulating hemoglobin oxygen affinity (BPGM) (16,17).…”

A new HPLC-based assay for the measurement of fructosamine-3-kinase (FN3K) and FN3K-related protein activity in human erythrocytes

“…Amadori compounds such as fructosamines are formed during the early stage of glycation and represent the main products. This protein modification may lead to altered function, e.g., decreased activity of 2,3-bisphosphoglycerate mutase [1]. The two ketosamine kinases, fructosamine-3-kinase (FN3K) and fructosamine-3-kinase-related protein (FN3K-RP), can deglycate free and protein-bound Amadori compounds and are thus considered as protein repair enzymes [2].…”