The Ehlers-Danlos syndrome (EDS) is a heterogeneous group of connective tissue disorders affecting skin and joint function. Molecular defects in extracellular matrix proteins, including collagen (type I, III, and V) and tenascin X are associated with different forms of EDS. Compound heterozygous mutations in the B4GALT7 gene, resulting in aberrant glycosylation of the dermatan sulfate proteoglycan decorin, had been described in a single patient affected with the progeroid form of EDS. We have studied the molecular phenotype of decorin, biglycan, and collagen type I containing fibrils in skin fibroblasts of a patient carrying the novel homozygous C808T point mutation in the B4GALT7 gene, which causes an Arg270Cys substitution in beta4GalT-7. Compared to control fibroblasts, galactosyltransferase activity in beta4GalT-7(Arg270Cys) cells was approximately three times reduced over a temperature range of 25-41 degrees C. Pulse-chase experiments and confocal microscopy demonstrated that synthesis and secretion of decorin were normal in beta4GalT-7(Arg270Cys) cells. However, about 50% of decorin were synthesized as a protein core in addition to its proteoglycan form. Biglycan was found in a monoglycanated form in addition to its mature form. Glycosaminoglycan chains were of the dermatan/chondroitin sulfate type both in beta4GalT-7(Arg270Cys) and control cells, and epimerization was reduced for decorin and biglycan. Compared to control cells, beta4GalT-7(Arg270Cys) cells showed altered, highly spread or stretched phenotypes and decreased proliferation rates. At the ultrastructural level, an intracellular accumulation of multiple secondary lysosomes and degenerative vacuoles was seen in beta4GalT-7(Arg270Cys) cells. Furthermore, the collagen suprastructures were altered in the beta4GalT-7(Arg270Cys) cells. The reduced beta4GalT-7 activity resulting in defective glycosylation of decorin and biglycan may be responsible for the complex molecular pathology in beta4GalT-7 deficient EDS patients, given the role of these proteoglycans in bone formation, collagen fibrillogenesis, and skeletal muscle development.