Abnormal Galactosylation of Complex Carbohydrates in Cultured Fibroblasts from Patients with Galactose-1-Phosphate Uridyltransferase Deficiency

Abstract

ABSTRACT. An abnormality in galactosylation of complex carbohydrates may be important in the pathogenesis of the long-term complications of classic (galactose-lphosphate uridyltransferase-deficient) galactosemia. The ability of nine galactosemic fibroblast preparations to be galactosylated with a purified galactosyltransferase was measured as an indicator of vacant sites where galactose would normally reside. The amount of galactose transferred to cell protein from galactosemic patients was significantly higher than that transferred to a group of seven controls (p < 0.005). Galactosyltransferase activity of the galactosemic cell preparation toward N-acetylglucosamine was also significantly higher than normal (p < 0.01), and there was a linear relationship between these two parameters in galactosemic but not normal cells. These findings suggest that there is defective galactosylation of galactosemic cell complex carbohydrates and that such cells increase their galactosyltransferase levels in an attempt to compensate for the defect. Defective galactosylation may be implicated as an etiologic factor in complications observed in galactosemic patients even when treated with galactose-restricted diets. (Pediatr Res 31: 508-511, 1992) Abbreviations UDPgalactose, uridine diphosphogalactose UDPglucose, uridine diphosphoglucose OPCA, olivopontocerebellar atrophy Although dietary restriction of galactose has been the basis for treatment of classical galactosemia for over 50 years (I), the longterm efficacy of this approach has been seriously questioned. A survey of over 300 patients indicates that even well-treated patients whose galactose restriction was initiated at birth have developmental delay, speech abnormalities, and ovarian failure in females (2). Older galactose-restricted patients have developed an ataxia syndrome (3).Two theories have been proposed, both involving the sugar nucleotide UDPgalactose, to explain the presence of the complications seen despite the absence of dietary galactose. Gitzelmann et al. (4) (7) recently found that the galactose to mannose ratio is abnormally low in a hydrolysate of the cultured fibroblasts of galactosemic patients, supporting the idea of defective galactosylation in these cells. To test the latter hypothesis, we studied the galactosylation state of glycoproteins in cultured fibroblasts by determining the extent to which cellular glycoproteins would serve as galactose acceptors when extracts were incubated with a purified galactosyltransferase. The rationale for this approach was that an impairment of galactosylation would result in a greater number of glycoprotein oligosaccharides being terminated in N-acetylglucosamine. Normally, galactose is linked to N-acetylglucosamine in the biosynthesis of the carbohydrate moieties of glycoproteins by a galactosyltransferase (Fig. 1). We reasoned that if we incubated cell extracts with radiolabeled UDPgalactose and purified galactosyltransferase, radioactive galactose would be transferred to terminal N-acetylglucosamine sites (Fig. 1)....

Involvement of endoplasmic reticulum stress in a novel Classic Galactosemia model

“…Since UDP-glucose/-galactose are crucial glycosyl donors, their deficiencies in these cells will undoubtedly impair protein glycosylation reactions [50], and could in turn trigger ER stress [30,31,50]. Indeed, aberrant glycosylation of glycoproteins and glycolipids have been widely reported in patients with Classic Galactosemia [13,[51][52][53][54][55]. The reduction of EGFR, a heavily glycosylated protein with 11 Nlinked glycosylation sites in the GALT-deficient cells observed in this study is likely to be resulted from impaired protein glycosylation, which would have initiated ER stress in this case.…”

The molecular basis of galactosemia — Past, present and future

“…Defects in the glycosylation of neuronal cells from a galactosemic patient was first noted in the early 1970s (Haberland et al , A C C E P T E D M A N U S C R I P T 14 1971). Reduced levels of galactosylation of proteins from cells and serum derived from patients with type I galactosemia has been observed in several studies (Dobbie et al , 1990;Ornstein et al , 1992;Stibler et al , 1997;Charlwood et al , 1998;Coss et al , 2014). It was hypothesised that this is caused by the reduced levels of UDP-galactose often observed in cells derived from galactosemia patients (W. G. Ng et al , 1989).…”

Classical Galactosaemia and CDG, the N-Glycosylation Interface. A Review

“…These intermediates can result in competitive inhibition of glycosyltransferases (Lai et al 2003). A shortage of endproduct UDP-hexose sugars could also lead to disruption of glycosylation in the posttranslational modification (PTM) of proteins and lipids (Ng et al 1989;Ornstein et al 1992).…”