Regulation and localization of cellular retinol-binding protein, retinol-binding protein, cellular retinoic acid-binding protein (CRABP), and CRABP II in the uterus of the pseudopregnant rat.

Three members of the superfamily of small intracellular carrier proteins for lipophilic compounds are cellular retinol-binding protein (CRBP), cellular retinoic acid-binding protein (CRABP), and cellular retinoic acid-binding protein II (CRABP II). Retinol-binding protein (RBP) is a secreted protein that binds and solubilizes vitamin A for transport. Here we report the coordinate regulation of RBP, CRBP, retinol, and CRABP II in the uterus of the pseudopregnant rat. In the proliferative stage of the uterus, which was induced by PMSG, the messenger RNA (mRNA) and protein levels of RBP and CRBP as well as retinol levels significantly decreased. This pattern of regulation was duplicated by estrogen treatment of prepubertal rats. In addition, CRBP and RBP were found to be colocalized to the stromal cells of the rat uterus by immunohistochemistry and [35S]methionine-labeled affinity chromatography, respectively, and were not detected in other cell populations. CRABP II mRNA and protein expression were up-regulated in the proliferative phase of the uterus brought about by PMSG injection or, alternatively, by estrogen treatment of prepubertal rats. CRABP II was localized to the surface epithelium, but was not seen elsewhere, including glandular epithelium. Immunolocalization of CRABP showed staining of the smooth muscle and stromal cells of the uterus. The appearance of CRABP in the stroma of the uterus also correlated with PMSG injection as well as estrogen treatment. Although estrogen induced the appearance of both binding proteins, CRABP mRNA levels peaked between 4-24 h postestrogen treatment, whereas CRABP II mRNA levels continued to rise 48 h postestrogen treatment. These data demonstrate an important role for vitamin A and retinoid-binding proteins in rat uterine physiology.

Test of the contribution of an amino-aromatic hydrogen bond to protein function.

Hydrogen bonds which form between a hydrogen bond donor and an aromatic ring as acceptor are thought to contribute to the stability and function of proteins. We have tested the function of such an interaction in a highly homologous pair of proteins, cellular retinol-binding protein (CRBP) and cellular retinol-binding protein, type II [CRBP(II)]. Both proteins bind the ligand all-trans-retinal with comparable affinities, but CRBP has an approximately 100-fold higher affinity for all-trans retinal. The greater affinity of CRBP for all-trans-retinol has been attributed to the presence of an amino-aromatic hydrogen bond, which is absent in CRBP(II). We have generated a pair of mutant proteins, in which the amino-aromatic interaction was removed from CRBP and introduced into CRBP(II). Spectral analyses of retinol when bound to the wild-type and mutant CRBP suggested that it adopted an identical conformation within both proteins, a conformation that was distinct from that of retinol bound to CRBP(II), both wild-type and mutant. Unexpectedly, the affinities of the mutant binding proteins for all-trans-retinol were indistinguishable from those of their corresponding wild-type proteins. Further, in ligand competition experiments, there were no observable differences between mutant and wild-type CRBP, or between mutant and wild-type CRBP(II), in their preferences for binding all-trans-retinol versus all-trans-retinal. The results of this direct test of the proposed function of an amino-aromatic hydrogen bond did not support a functional role for such bonds, at least in this system.

Purification and partial characterization of a retinyl ester hydrolase from the brush border of rat small intestine mucosa: probable identity with brush border phospholipase B.

Retinol esterified with long-chain fatty acids is a common dietary source of vitamin A, that is hydrolyzed prior to absorption. An intrinsic brush border membrane retinyl ester hydrolase activity had previously been demonstrated for rat small intestine [Rigtrup, K. M., & Ong, D. E. (1992) Biochemistry 31, 2920-2926]. This activity has now been purified to apparent homogeneity by a three-column procedure to obtain a protein of apparent molecular weight of 130,000. The purified protein retained the pattern of bile salt stimulation, specificity for the acyl moiety of the retinyl ester, and the Km values previously observed for the activity present in the isolated brush border membrane. This protein also had a potent phospholipase activity, while having little measurable ability to hydrolyze triacylglyceride and cholesteryl ester substrates. The retinyl ester hydrolase enzyme was localized to the distal two-thirds of the small intestine. A polyclonal antiserum against rat brush border phospholipase B reacted with the purified retinyl ester hydrolase, strongly suggesting that this enzyme was the same as that previously purified and characterized as a calcium-independent brush border phospholipase B [Pind, S., & Kuksis, A. (1991) Biochem. Cell Biol. 69, 346-357]. Detailed kinetic studies revealed lower Km values for retinyl palmitate substrate compared to phosphatidylcholine substrate, with all tested bile salts. The Km values for each substrate were bile salt dependent and differently altered when bile salts were changed. Vmax values were also bile salt dependent. Retinyl palmitate was hydrolyzed most rapidly in the presence of deoxycholate and least rapidly in taurocholate.(ABSTRACT TRUNCATED AT 250 WORDS)

Intestinal vitamin A metabolism: coordinate distribution of enzymes and CRBP(II).

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.

Ontogeny of two vitamin A-metabolizing enzymes and two retinol-binding proteins present in the small intestine of the rat.

The patterns of expression of cellular retinol-binding protein (CRBP), cellular retinol-binding protein, type two [CRBP(II)], lecithin: retinol acyltransferase (LRAT), and microsomal retinal reductase were examined for rat small intestine during the perinatal period. CRBP was present (15 pmole per mg soluble protein) at the earliest time examined, the 16th day of gestation, declining by 70% by birth, maintained to adulthood. In contrast, CRBP(II) appeared 2-3 days before birth, rising to its highest level (500 pmole per mg soluble protein) by day 3 after birth, then declining by 50% during the late suckling period to the adult level. Immunohistochemistry revealed that CRBP(II) initially appeared in the epithelial cell layer in a patchy manner, resolving by birth into an even staining of all villus-associated enterocytes. In contrast, CRBP was evenly expressed in the epithelial cell layer at day 17/18 but was absent by birth. Intestinal LRAT activity increased rapidly in the 2 days prior to birth, then declined at weaning to the adult level. Microsomal retinal reductase was measurable in the intestine at birth, but not detected during the early suckling period, reappearing at day 21. Considerable increase was then observed coincident with weaning, when carotenes, from which retinal is derived, became an important source of vitamin A. The pattern of appearance of these elements appears to prepare the intestine for the necessary processing of vitamin A required after birth.

Purification, primary structure characterization, and cellular distribution of two forms of cellular retinol-binding protein, type II from adult rat small intestine.

Cellular retinol-binding protein type II (CRBP(II)) is a major protein in the small intestine, accounting for more than 1% of the soluble protein recovered from rat jejunal mucosa. Two forms of the protein, called CRBP(II)A and CRBP(II)B, were purified from rat small intestine using a three-column procedure. The two forms were present in equal abundance. The primary structures of CRBP(II)A and CRBP(II)B were determined using a combination of techniques including amino acid composition and sequence analyses, and fast atom bombardment and gas chromatography-electron impact mass spectrometry. The primary structures of both proteins were found to be identical, but they differed in their NH2-terminal processing. CRBP(II)B was acetylated at its NH2 terminus, while CRBP(II)A was not. The results also confirmed the amino acid sequence of CRBP(II)A that was deduced from the cDNA sequence by Li et al. (Li, E., Demmer, L. A., Sweetser, D. A., Ong, D. E., and Gordon, J. I. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5770-5783). Antibodies capable of distinguishing between the two forms of CRBP(II) were used for immunohistochemical studies which indicated that the organ and cellular distributions of the two forms were identical. The 50% acetylation observed here in vivo fits the pattern predicted by recent in vitro studies which described the effect of NH2-terminal sequence on cotranslational NH2-terminal processing of cytosolic proteins (Boissel, J. P., Kasper, T. J., and Bunn, H. F. (1988) J. Biol. Chem. 263, 8443-8449). Our results provide a basis for investigating the possibility of different roles of CRBP(II)A and CRBP(II)B within cells, as well as the importance of acetylation of the amino terminus for these biological functions.

Reduction of retinaldehyde bound to cellular retinol-binding protein (type II) by microsomes from rat small intestine.

Cellular retinol-binding protein, type II (CRBP (II], an abundant protein of the rat small intestine, has recently been shown to be able to bind retinaldehyde in addition to retinol (MacDonald, P.N., and Ong, D. E. (1987) J. Biol. Chem. 262, 10550-10556). Retinaldehyde is produced in the intestine by oxidative cleavage of beta-carotene. The next step in the intestinal metabolism of vitamin A is the reduction of retinaldehyde to retinol which is then esterified for incorporation into chylomicrons. In the present study retinaldehyde bound to CRBP(II) was found to be available for reduction by microsomal preparations from rat small intestinal mucosa. The microsomal activity was about 8 times greater than the activity observed for an equal amount of cytosolic protein. Retinaldehyde reduction utilized either NADH or NADPH as cofactor, with NADH being slightly more effective. The apparent Km for retinaldehyde-CRBP(II) was 0.5 microM, and the Vmax was approximately 300 pmol/min/mg protein, a rate more than sufficient for the needs of the animal. The product retinol remained complexed to CRBP(II). The microsomal enzyme activity reduced free and bound retinaldehyde to approximately the same extent, although the aldehyde function of retinaldehyde bound to CRBP(II) was less accessible to chemical reducing agents than that of free retinaldehyde. Retinol bound to CRBP(II) could not be oxidized by the microsomal activity in the presence of NAD+, while free retinol or retinol bound to bovine serum albumin was oxidized to retinaldehyde. The more favorable reduction versus oxidation of retinoid bound to CRBP(II) consequently favored the reaction known to be required for the ultimate conversion of beta-carotene to retinyl esters for export from the gut.

Acyl-CoA-independent esterification of retinol bound to cellular retinol-binding protein (type II) by microsomes from rat small intestine.

Cellular retinol-binding protein (type II) (CRBP(II)), a newly described retinol-binding protein, is present in the small intestinal absorptive cell at high levels. Retinol (vitamin A alcohol) presented as a complex with CRBP(II) was found here to be esterified by microsomal preparations from rat small intestinal mucosa. The esterification observed utilized an endogenous acyl donor(s) and produced retinyl esters containing linoleate, oleate, palmitate, and stearate in a proportion quite similar to that previously reported for retinyl esters in lymph and isolated chylomicrons of rat. No dependence on endogenous or exogenous acyl-CoA could be demonstrated. The apparent Km for retinol-CRBP(II) in the reaction with endogenous acyl donor was 2.4 X 10(-7) M. Retinol presented as a complex with CRBP(II) was esterified more than retinol presented as a complex with cellular retinol-binding protein or retinol-binding protein, two other proteins known to bind retinol in vivo, but about the same as retinol presented bound to bovine serum albumin or beta-lactoglobulin. The ability of protein-bound retinol to be esterified was related to accessibility of the hydroxyl group, as judged by the ability of alcohol dehydrogenase to oxidize the bound retinol. However, whereas retinol bound to CRBP(II) was unavailable for esterification in any acyl-CoA-dependent reaction, retinol bound to bovine serum albumin was rapidly esterified in a reaction utilizing exogenous acyl-CoA. The results suggest that one of the functions of CRBP(II) is to accept retinol after it is absorbed or generated from carotenes in the small intestine and present it to the appropriate esterifying enzyme.

Perinatal rat lung retinol (vitamin A) and retinyl palmitate.

The potential role for retinol (vitamin A alcohol) in the differentiation of the developing lung prompted this study in the perinatal rat. High performance liquid chromatography was used to separate, detect, and quantitate retinol and retinyl palmitate in lipid extracts of tissue and serum. Fetal and maternal blood showed the presence of retinol, whereas no retinyl palmitate was detected. On the other hand, fetal and postnatal lungs contained retinyl palmitate as well as retinol. Considerable changes in the content of lung retinyl palmitate were found during lung development. Fetal lungs (17-21 days of gestation) contained 2.3 +/- 0.36 micrograms/g wet weight (mean +/- SD) of retinyl palmitate and 0.14 +/- 0.05 micrograms/g of retinol. Lungs of pups (1-10 days old) contained much less retinyl palmitate, 0.63 +/- 0.20 micrograms/g, whereas the amount of retinol was the same as in fetal lungs. The surprisingly high content of retinyl palmitate in fetal lung and its depletion after birth may be functionally related to retinol action in the developing lung.

Cross-linking and chymotryptic digestion of the extracytoplasmic domain of the anion exchange channel in intact human erythrocytes.

We have applied our new high yield, membrane-impermeant, protein cross-linking reagents (J.V. Staros, 1982. Biochemistry 21:3950-3955) together with chymotryptic digestion of the surface of intact erythrocytes (T.L. Steck, B. Ramos, and E. Strapazon, 1976. Biochemistry 15:1154-1161) in an investigation of the topology of the extracytoplasmic domain of the anion exchange channel of intact human erythrocytes. In intact erythrocytes, these cross-linking reagents have been shown to cross-link subunits of the anion exchange channel to dimers in the extracytoplasmic domain of the protein. Chymotryptic treatment of intact erythrocytes has been shown to cleave subunits of the anion exchange channel into two fragments of distinct Mr. Sequential treatment of intact erythrocytes with either of two membrane-impermeant cross-linkers, followed by digestion with chymotrypsin, yields chymotryptic fragments of the anion exchange channel cross-linked to one another. The cross-linked products observed appear to arise by cross-linking of unlike chymotryptic fragments, whether the cross-links are intersubunit or intrasubunit. These results are consistent with a model of the anion exchange channel in which the subunits form a head-to-head dimer with a twofold center of symmetry perpendicular to the plane of the membrane.