Evidence for a Terminal α-d-Galactopyranosyl Residue in Galactosylgalactosylglucosylceramide from Human Kidney

Galactosylgalactosylglucosylceramide (CTH) was extracted and purified from pooled normal human kidney tissue and also from the kidney of a patient with Fabry's disease, a glycosphingolipid storage disease characterized by a defect in the catabolism of CTH. Nuclear magnetic resonance spectra of the intact molecules were recorded at 100 and 220 MHz in pyridine-d5 at various temperatures. The characteristics of signals attributable to the anomeric protons are consistent with the following structural features: a β- d -glucopyranosyl residue linked to ceramide (H-1, doublet, δ = 4.74 ppm, J = 7.6 Hz);an internal, β- d -galactopyranosyl residue (H-1, doublet, δ = 4.94 ppm, J = 7.5 Hz);a terminal α- d -galactopyranosyl residue (H-1, doublet, δ = 5.45 ppm, J = 4.1 Hz). Optical rotatory data are also consistent with such a formulation for the glycolipid. CTH and lactosylceramide labeled in the terminal d -galactosyl moiety were prepared by oxidation with the enzyme d -galactose oxidase (EC 1.1.3.-) and reduction of the resulting hexodialdoses with [3H]NaBH4. With the use of these substrates, it was found that the terminal d -galactosidic linkage of CTH, but not of lactosylceramide, was hydrolyzed by partially purified α- d -galactoside galactohydrolase (EC 3.2.1.22) from green coffee beans. These physicochemical and enzymic data are presented as proof that the terminal d -galactopyranosyl residue of the CTH of human renal tissue has the α stereoconfiguration.