The biotin cycle and the HCS-dependent transcriptional regulation in human cells. Shown is a schematic representation of the biotin cycle (green arrows) and the HCS-dependent signal transduction pathway (red arrows) responsible for the transcriptional regulation of PC, PCC, MCC, ACC, sodium-dependent multivitamin transporter (SMVT), and HCS. The enzymes participating in the HCS pathway are sGC and PKG.

Effect of biocytin (biotinyl-lysine) and biotin concentration in carboxylase biotinylation of normal and MCD-BD fibroblasts cultivated in biotin-deficient medium. Normal (A and B) and MCD-BD fibroblasts (C and D) were incubated in biotin-deficient medium for 13 days (biotin deficient cell) to reduce the amount of biotin present in carboxylases. The cells were then transferred to a medium containing 1, 10, or 100 nm of biotin (A and C) or biocytin (B and D) for 24 h. Recovery of carboxylase biotinylation was determined by densitometric analysis of the streptavidin-AP reactive Western blot MCC/PCC band, as described under “Experimental Procedures.” Representative Western blots are shown above their corresponding histogram bars. The data were normalized with respect to the MCC/PCC biotin content of each cell line grown in biotin-replete medium (control) and shown as the means ± S.E. of three independent experiments.

Time course of the effect of biotin deficiency on holocarboxylase synthetase, biotinidase, and β-actin mRNA levels in normal and MCD-BD fibroblasts. Normal (A) and MCD-BD cells (B) were grown in biotin-deficient medium for up to 13 days, and the mRNA levels for HCS (•), biotinidase (▴), and β-actin (▪) were determined by reverse transcription-PCR analysis at various times, as described under “Experimental Procedures.” The results are presented as percentages of mRNA values in cells grown in biotin-replete medium and normalized to initial (time 0) β-actin mRNA levels. The data are from three different experiments and shown as the means ± S.E.

Molecular and functional characterization of cell line MCD-BD. A, partial biotinidase amino acid sequence (residues 140–180 and 420–460) localizing substitutions A171T and D444H found in the cell line MCD-BD. The corresponding nucleotide sequence is shown above the amino acid sequences with mutations indicated in bold characters. B, representative Western blot experiment showing the effect of biotinidase deficiency (MCD-BD cells) in the biotinylation of PC, MCC, and PCC following growth in biotin replete (0.40 μm biotin) medium. The MCD-MK cells are from a patient with HCS deficiency (3). The changes in streptavidin-AP-reactive MCC/PCC bands were analyzed by densitometry. The values below the lanes correspond to the relative densitometry value (RDV) normalized to the value observed in normal fibroblasts. Each lane contains 50 μg of crude cell lysate.

Effect of biotin, ODQ and 8-Br-cGMP on carboxylase biotinylation and PC and MCC protein levels in normal and MCD-BD fibroblasts. Normal (A, C, and E) and MCD-BD fibroblasts (B, D, and F) were grown in normal biotin containing medium (N) or biotin-free medium (B def) for 13 days. After this period the cells were stimulated with 1 μm biotin for 48 or 72 h. A and B, cell extracts were prepared from the cell cultures, and the status of carboxylases biotinylation was determined by Western blot using streptavidin-AP as described under “Experimental Procedures.” C and D, the effect biotin deficiency and restoration of PC and MCC protein levels was determined by Western blot using polyclonal antibodies directed against these carboxylases. E, to explore the role of the HCS-sGC-PKG pathways on the biotin-dependent restoration of MCC protein levels, we studied the effect of inhibiting sGC on biotin-starved normal fibroblasts stimulated with 1 μm biotin. Alternatively, biotin-starved MCD-BD fibroblasts were stimulated with 1 μm biotin or 8-Br-cGMP, and the levels of MCC protein determined by Western blot analysis as previously described. To verify that differences in biotinylation or protein levels were not related to the amount of protein present in the samples, all of the gels were Coomassie-stained before transferring of the proteins to membranes. An example of these loading controls is depicted in A under streptavidin-AP bands.

Holocarboxylase synthetase activity and protein in MCD-BD cells. HCS activity was determined in a solid phase assay in which p67 bound to PVDF membrane was used as substrate for biotinylation. A, preparation of p67 substrate. A PVDF membrane was prepared by transfer from a polyacrylamide gel following electrophoresis of extracts of E. coli XL-1 (wild type) or C-124 cells (biotin auxotroph) expressing p67. The two protein bands correspond to BCCP (upper band, 18 kDa) and p67 (lower band, 14 kDa). The membrane was developed with streptavidin-AP (left two lanes) to detect biotinylated proteins or anti-FLAG antibody (right lane) to detect unbiotinylated p67. In XL-1 cells both bands are biotinylated because of endogenously synthesized biotin. In C-124 cells, in which p67 expression was induced in biotin-free medium, only the upper band is biotinylated. The lane on the right confirms the presence of unbiotinylated p67 in the C-124 cells using the FLAG antibody. B, HCS activity of fibroblast cultures. PVDF membrane-bound p67 was incubated in the presence of [³H]biotin, ATP, and total protein extracts from human fibroblasts. The amount of [³H]biotin incorporated in p67 by normal fibroblasts (white bar) or MCD-BD cells (black bar) was determined using a liquid scintillation counter. Differences between normal and MCD-BD cells shown to be statistically significant (p < 0.05). C, detection of HCS protein in fibroblast cultures. The level of HCS protein in normal fibroblasts and MCD-BD cells was visualized by Western blot using an antibody against the amino-terminal region of HCS.