Identification of ABC transporters acting in vitamin B12 metabolism in Caenorhabditis elegans.

Vitamin B12 (cobalamin, Cbl) is a micronutrient essential to human health. Cbl is not utilized as is but must go through complex subcellular and metabolic processing to generate two cofactor forms: methyl-Cbl for methionine synthase, a cytosolic enzyme; and adenosyl-Cbl for methylmalonyl-CoA mutase, a mitochondrial enzyme. Some 10-12 human genes have been identified responsible for the intracellular conversion of Cbl to cofactor forms, including genes that code for ATP-binding cassette (ABC) transporters acting at the lysosomal and plasma membranes. However, the gene for mitochondrial uptake is not known. We hypothesized that ABC transporters should be candidates for other uptake and efflux functions, including mitochondrial transport, and set out to screen ABC transporter mutants for blocks in Cbl utilization using the nematode roundworm Caenorhabditis elegans. Thirty-seven mutant ABC transporters were screened for the excretion of methylmalonic acid (MMA), which should result from loss of Cbl transport into the mitochondria. One mutant, wht-6, showed elevated MMA excretion and reduced [14C]-propionate incorporation, pointing to a functional block in methylmalonyl-CoA mutase. In contrast, the wht-6 mutant appeared to have a normal cytosolic pathway based on analysis of cystathionine excretion, suggesting that cytosolic methionine synthase was functioning properly. Further, the MMA excretion in wht-6 could be partially reversed by including vitamin B12 in the assay medium. The human ortholog of wht-6 is a member of the G family of ABC transporters. We propose wht-6 as a candidate for the transport of Cbl into mitochondria and suggest that a member of the corresponding ABCG family of ABC transporters has this role in humans. Our ABC transporter screen also revealed that mrp-1 and mrp-2 mutants excreted lower MMA than wild type, suggesting they were concentrating intracellular Cbl, consistent with the cellular efflux defect proposed for the mammalian MRP1 ABC transporter.

Structural impact of human and Escherichia coli biotin carboxyl carrier proteins on biotin attachment.

Holocarboxylase synthetase (HCS, human) and BirA (Escherichia coli) are biotin protein ligases that catalyze the ATP-dependent attachment of biotin to apocarboxylases. Biotin attachment occurs on a highly conserved lysine residue within a consensus sequence (Ala/Val-Met-Lys-Met) that is found in carboxylases in most organisms. Numerous studies have indicated that HCS and BirA, as well as biotin protein ligases from other organisms, can attach biotin to apocarboxylases from different organisms, indicating that the mechanism of biotin attachment is well conserved. In this study, we examined the cross-reactivity of biotin attachment between human and bacterial biotin ligases by comparing biotinylation of p-67 and BCCP87, the biotin-attachment domain fragments from human propionyl-CoA carboxylase and E. coli acetyl-CoA carboxylase, respectively. While BirA has similar biotinylation activity toward the two substrates, HCS has reduced activity toward bacterial BCCP87 relative to its native substrate, p-67. The crystal structure of a digested form of p-67, spanning a sequence that contains a seven-residue protruding thumb loop in BCCP87, revealed the absence of a similar structure in the human peptide. Significantly, an engineered "thumbless" bacterial BCCP87 could be biotinylated by HCS, with substrate affinity restored to near normal. This study suggests that the thumb loop found in bacterial carboxylases interferes with optimal interaction with the mammalian biotin protein ligase. While the function of the thumb loop remains unknown, these results indicate a constraint on specificity of the bacterial substrate for biotin attachment that is not itself a feature of BirA.

Biotin is not a natural histone modification.

In addition to its role as the cofactor of biotin-dependent carboxylases, biotin has been demonstrated to have a role in cellular processes including transcription and gene silencing. Histones have been proposed to be modified by biotin in a site-specific manner, providing a pathway by which biotin acts as a regulatory molecule for gene expression. However, there is uncertainty whether biotin attachment to histones in vitro can be extrapolated to biotin as a native histone modification. We critically examined a number of methods used to detect biotin attachment on histones, including [(3)H]-biotin uptake, Western blot analysis of histones, and mass spectrometry of affinity purified histone fragments with the objective of determining if the in vivo occurrence of histone biotinylation could be conclusively established. We found for each of these methods that, while biotin could be readily detected on native carboxylases or histones biotinylated in vitro, biotin attachment on native histones could not be detected in cell cultures from various sources. We conclude that biotin is absent in native histones to a sensitivity of at least one part per 100,000, suggesting that the regulatory impact of biotin on gene expression must be through alternate mechanisms.

A genome-based functional screening approach to vaccine development that combines in vitro assays and DNA immunization.

A two-step screening strategy was developed to identify strong immunogenic polypeptides with putative vaccine and/or adjuvant activity. In the first step, a mycobacterial genomic DNA library was screened in vitro to identify plasmids encoding polypeptides that stimulate splenocytes from mycobacteria-immunized mice and T cells from PPD-positive healthy donors to produce interferon-gamma. In the second step, plasmids were selected for their ability to induce protective immunity in a mouse model of tuberculosis following DNA immunization. The potential of this approach is illustrated by the identification of a panel of immunogenic polypeptides that may be used to engineer a new generation of vaccines.