RESUMEN
Cellular retinol-binding proteins types I and II (CRBP-I and CRBP-II) are known to differentially facilitate retinoid metabolism by several membrane-associated enzymes. The mechanism of ligand transfer to phospholipid small unilamellar vesicles was compared in order to determine whether differences in ligand trafficking properties could underlie these functional differences. Unidirectional transfer of retinol from the CRBPs to membranes was monitored by following the increase in intrinsic protein fluorescence that occurs upon ligand dissociation. The results showed that ligand transfer of retinol from CRBP-I was >5-fold faster than transfer from CRBP-II. For both proteins, transfer of the other naturally occurring retinoid, retinaldehyde, was 4-5-fold faster than transfer of retinol. Rates of ligand transfer from CRBP-I to small unilamellar vesicles increased with increasing concentration of acceptor membrane and with the incorporation of the anionic lipids cardiolipin or phosphatidylserine into membranes. In contrast, transfer from CRBP-II was unaffected by either membrane concentration or composition. Preincubation of anionic vesicles with CRBP-I was able to prevent cytochrome c, a peripheral membrane protein, from binding, whereas CRBP-II was ineffective. In addition, monolayer exclusion experiments demonstrated differences in the rate and magnitude of the CRBP interactions with phospholipid membranes. These results suggest that the mechanisms of ligand transfer from CRBP-I and CRBP-II to membranes are markedly different as follows: transfer from CRBP-I may involve and require effective collisional interactions with membranes, whereas a diffusional process primarily mediates transfer from CRBP-II. These differences may help account for their distinct functional roles in the modulation of intracellular retinoid metabolism.
Asunto(s)
Lípidos de la Membrana/metabolismo , Fosfolípidos/metabolismo , Retinoides/metabolismo , Proteínas de Unión al Retinol/metabolismo , Adsorción , Transporte Biológico , Grupo Citocromo c/metabolismo , Cinética , Modelos Moleculares , Concentración Osmolar , Retinaldehído/metabolismo , Proteínas Celulares de Unión al Retinol , Vitamina A/metabolismoRESUMEN
The structure of heart fatty acid binding protein (HFABP) is a flattened beta-barrel comprising 10 antiparallel beta-sheets capped by two alpha-helical segments. The helical cap region is hypothesized to behave as a portal "lid" for the entry and release of ligand from the binding pocket. The transfer of fatty acid from HFABP is thought to occur via effective collisional interactions with membranes, and these interactions are enhanced when transfer is to membranes of net negative charge, thus implying that specific basic residues on the surface of HFABP may govern the transfer process [Wootan, M. G., & Storch, J. (1994) J. Biol. Chem. 269, 10517-10523]. To directly examine the role of charged lysine residues on the HFABP surface in specific interactions with membranes, chemical modification and selective mutagenesis of HFABP were used. All surface lysine residues were neutralized by acetylation of recombinant HFABP with acetic anhydride. In addition, seven mutant HFABPs were generated that resulted in charge alterations in five distinct sites of HFABP. Modification of the protein did not significantly alter the structural or ligand binding properties of HFABP, as assessed by circular dichroism, fluorescence quantum yield, and ligand binding analyses. By using a resonance energy transfer assay, transfer of 2-(9-anthroyloxy)palmitate (2AP) from acetylated HFABP to membranes was significantly slower than transfer from native HFABP. In addition, in distinct contrast to transfer from native protein, the 2AP transfer rate from acetylated HFABP was not increased to acceptor membranes of increased negative charge. Transfer of 2AP from HFABP mutants involving K22, located on alpha-helix I (alpha-I) of the helical cap region, was 3-fold slower than transfer from wild-type protein, whereas rates from a mutant involving the K59 residue, located on the beta 2-turn of the barrel near the helical cap, were 2-fold faster than those of wild type. A double mutant involving K22 and K59 resulted in transfer rates identical to those of wild type, indicating that at least two domains are involved in determining the overall rate of ligand transfer. In addition, 2AP transfer rates from HFABP mutated at position 22 were totally unaffected by the charge characteristics of acceptor membranes, in marked contrast to wild type and other members of the mutant series. Further, by introducing a positive charge to alpha-helix II (alpha-II) of the helical cap region, 2AP transfer rates increased by 4-fold and properties of HFABP transfer began to approach those seen for AFABP, another member of the FABP family thought to transfer ligand via collisional interactions with membranes, which has a lysine residue in the alpha-II helix. These studies demonstrate that the helical cap region of HFABP may play an important role in governing ionic interactions between binding protein and membranes.
Asunto(s)
Proteínas Portadoras/metabolismo , Membranas Artificiales , Proteína P2 de Mielina/metabolismo , Proteínas de Neoplasias , Proteínas del Tejido Nervioso , Fosfolípidos/metabolismo , Acetilación , Animales , Secuencia de Bases , Proteínas Portadoras/genética , Dicroismo Circular , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Cinética , Ligandos , Lisina/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteína P2 de Mielina/genética , Concentración Osmolar , Ácidos Palmíticos/metabolismo , Unión Proteica , Conformación Proteica , Ratas , Proteínas Recombinantes/metabolismo , Espectrometría de FluorescenciaRESUMEN
The transfer of unesterified fatty acids (FA) from adipocyte fatty acid binding protein (A-FABP) to phospholipid membranes is proposed to occur via a collisional mechanism involving transient ionic and hydrophobic interactions [Wootan & Storch (1994) J. Biol. Chem. 269, 10517-10523]. In particular, it was suggested that membrane acidic phospholipids might specifically interact with basic residues on the surface of A-FABP. Here we addressed whether lysine residues on the surface of the protein are involved in this collisional transfer mechanism. Recombinant A-FABP was acetylated to neutralize all positively charged surface lysine residues. Protein fluorescence, CD spectra, and chemical denaturant data indicate that acetylation did not substantially alter the conformational integrity of the protein, and nearly identical affinities were obtained for binding of the fluorescently labeled FA [12-(9-anthroyloxy)oleate] to native and acetylated protein. Transfer of 2-(9-anthroyloxy)palmitate (2AP) from acetylated A-FABP to small unilamellar vesicles (SUV) was 35-fold slower than from native protein. In addition, whereas the 2AP transfer rate from native A-FABP was directly dependent on SUV concentration, 2AP transfer from acetylated protein was independent on the concentration of acceptor membranes. Factors which alter aqueous-phase solubility of FA, such as ionic strength and acyl chain length and saturation, affected the AOFA transfer rate from acetylated but not native A-FABP. Finally, an increase in the negative charge density of the acceptor SUV resulted in a marked increase in the rate of transfer from native A-FABP but did not increase the rate from acetylated A-FABP.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Tejido Adiposo/metabolismo , Proteínas Portadoras/metabolismo , Ácidos Grasos/metabolismo , Lisina/metabolismo , Proteína P2 de Mielina/metabolismo , Proteínas de Neoplasias , Proteínas del Tejido Nervioso , Acetilación , Animales , Transporte Biológico Activo , Proteínas Portadoras/química , Electroquímica , Proteína de Unión a los Ácidos Grasos 7 , Proteínas de Unión a Ácidos Grasos , Colorantes Fluorescentes , Técnicas In Vitro , Membranas Intracelulares/metabolismo , Liposomas , Lisina/química , Ratones , Estructura Molecular , Proteína P2 de Mielina/química , Fosfolípidos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidad , Espectrometría de FluorescenciaRESUMEN
In the mucosal layer of the small intestine, we found nearly identical gradients of CRBP(II), retinal reductase, and LRAT levels down the duodenal-ileal axis, suggesting coordinate regulation of these three proteins. In all cases the level of binding protein or enzyme activity was greatest in the proximal intestine and then decreased sharply in the distal half. This pattern fits with the known capacity of the intestine to absorb vitamin A. In addition, the retinal reductase activity was found predominantly in the intestinal mucosa, while LRAT activity was found in both the intestinal mucosa and muscle. An even distribution of LRAT activity along the longitudinal axis of the intestinal muscle was consistent with an even distribution of CRBP in that tissue. In conjunction with LRAT activity and CRBP, we found endogenous retinyl ester stores in the intestinal muscle layer. The patterns of retinyl ester produced by LRAT in vitro and found in vivo were similar, with retinyl palmitate predominating and a high percentage comprised of retinyl stearate. We also observed a bile salt-independent retinyl ester hydrolase activity in intestinal muscle whose distribution paralleled the retinyl ester stores and LRAT levels. This hydrolase appears to be distinct from retinyl ester hydrolases described from other organs as its activity was insensitive to retinyl ester chain length, the presence of bile salts, or the addition of apo-CRBP. This activity was inhibited by diethyl-p-nitrophenyl-phosphate (IC50 100 microM) and diethylpyrocarbonate (IC50 10 microM), demonstrating a requirement for active serine and histidine residues. In addition, we describe an activity present in some intestinal microsomal preparations that can perturb determinations of reductase and LRAT activity and must be avoided.
Asunto(s)
Aciltransferasas/metabolismo , Oxidorreductasas de Alcohol/metabolismo , Intestino Delgado/metabolismo , Proteínas de Unión al Retinol/metabolismo , Vitamina A/metabolismo , Animales , Hidrolasas de Éster Carboxílico/metabolismo , Cromatografía Líquida de Alta Presión , Citosol/metabolismo , Mucosa Intestinal/metabolismo , Masculino , Microsomas/metabolismo , Músculo Liso/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Celulares de Unión al Retinol , Vitamina A/análogos & derivadosRESUMEN
Esterification of retinol (vitamin A alcohol) with long-chain fatty acids by lecithin-retinol acyltransferase (LRAT) is an important step in both the absorption and storage of vitamin A. Retinol in cells is bound by either cellular retinol binding protein (CRBP), present in most tissues including liver, or cellular retinol binding protein type II [CRBP(II)], present in the absorptive cell of the small intestine. Here we investigated whether retinol must dissociate from these carrier proteins in order to serve as a substrate for LRAT by comparing Michaelis constants for esterification of retinol presented either free or bound. Esterification of free retinol by both liver and intestinal LRAT resulted in Km values (0.63 and 0.44 microM, respectively) similar to those obtained for esterification of retinol-CRBP (0.20 and 0.78 microM, respectively) and esterification of retinol-CRBP(II) (0.24 and 0.32 microM, respectively). Because Kd values for retinol-CRBP and retinol-CRBP(II) are 10(-8)-10-(-10) M, these similar Km values indicated prior dissociation is not required and that direct binding protein-enzyme interaction must occur. Evidence for such interaction was obtained when apo-CRBP proved to be a potent competitive inhibitor of LRAT, with a KI (0.21 microM) lower than the Km for CRBP-retinol (0.78 microM). Apo-CRBP(II), in contrast, was a poor competitor for esterification of retinol bound to CRBP(II). Apo-CRBP reacted with 4 mM p-(chloromercuri)benzenesulfonic acid lost retinol binding ability but retained the ability to inhibit LRAT, confirming that the inhibition could not be explained by a reduction in the concentration of free retinol.(ABSTRACT TRUNCATED AT 250 WORDS)