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R A Reithmeier

Publications and source records attributed to R A Reithmeier.

At least 37 records · Page 2Linked to original sources
PubMed1996

Glycosylation of multiple extracytosolic loops in Band 3, a model polytopic membrane protein.

N-glycosylated sites in polytopic membrane proteins are usually localized to single extracytosolic (EC) loops containing more than 30 residues [Landolt-Marticorena and Reithmeier (1994) Biochem. J. 302, 253-260]. This may be due to a biosynthetic restriction whereby only a single loop of nascent polypeptide is available to the oligosaccharyl transferase in the lumen of the endoplasmic reticulum. To test this hypothesis, two types of N-glycosylation mutants were constructed using Band 3, a polytopic membrane protein that contains up to 14 transmembrane segments and a single endogenous site of N-glycosylation at Asn-642 in EC loop 4. In the first set of mutants, an additional N-glycosylation acceptor site (Asn-Xaa-Ser/Thr) was constructed by site-directed mutagenesis in EC loop 3, with or without retention of the endogenous site. In the second set of mutants, EC loop 4 was duplicated and inserted into EC loop 2, again with or without retention of the endogenous site. Cell-free translation experiments using reticulocyte lysates showed that microsomes were able to N-glycosylate multiple EC loops in these Band 3 mutants. The acceptor site in EC loop 3 was poorly N-glycosylated, probably due to the suboptimal size (25 residues) of this EC loop. The localization of N-glycosylation sites to single EC loops in multi-span membrane proteins is probably due to the absence of suitably positioned acceptor sites on multiple loops.

Amino Acid Sequence↗
PubMed1996

Structure of the band 3 transmembrane domain.

The N-glycosylated membrane domain of band 3 consists of multiple membrane spanning segments that come together to form a regulated transmembrane passage for the exchange of anions. In this article we review the structural features of the membrane domain of band 3. Electron microscopic analysis of 2-dimensional crystals have confirmed the dimeric nature of the protein and has provided the overall shape of the membrane domain. The high degree of sequence identity in the transmembrane segments, and the finding that these segments are helical and remain tightly associated after proteolytic cleavage of the connecting loops, suggests that the interactions between transmembrane helices are specific and form the foundation for the structure of the membrane domain. N-glycosylation of band 3 is not essential for the transport function of the protein. N-glycosylation mutagenesis indicates that band 3 can be glycosylated on multiple loops and spans the membrane 12 times. Red cell diseases (HEMPAS and SAO) that affect the band 3 oligosaccharide structure and other properties of the protein are the subject of continued studies.

Amino Acid Sequence↗
PubMed1995

Carbohydrate-deficient glycoprotein syndrome type II. An autosomal recessive N-acetylglucosaminyltransferase II deficiency different from typical hereditary erythroblastic multinuclearity, with a positive acidified-serum lysis test (HEMPAS).

Carbohydrate-deficient glycoprotein syndromes (CDGS) are a family of multisystemic congenital diseases resulting in underglycosylated glycoproteins, suggesting defective N-glycan assembly. Fibroblast extracts from two patients with a recently described variant of this disease (CDGS type II) have previously been shown to have over 98% reduced activity of UDP-GlcNAc:alpha-6-D-mannoside beta-1,2-N-acetylglucosaminyltransferase II [GlcNAc-TII; Jaeken, J., Schachter, H., Carchon, H., De Cock, P., Coddeville, B. & Spik, G. (1994) Arch. Dis. Childhood 71, 123-127]. We show in this paper that mononuclear cell extracts from one of these CDGS type-II patients have no detectable GlcNAc-TII activity and that similar extracts from 12 blood relatives of the patient, including his father, mother and brother, have GlcNAc-TII levels 32-67% that of normal levels (average 50.1% +/- 10.7% SD), consistent with an autosomal recessive disease. The poly(N-acetyllactosamine) content of erythrocyte membrane glycoproteins bands 3 and 4.5 of this CDGS patient were estimated, by tomato lectin blotting, to be reduced by 50% relative to samples obtained from blood relatives and normal controls. Similar to patients with hereditary erythroblastic multinuclearity with a positive acidified-serum lysis test (HEMPAS), erythrocyte membrane glycoproteins in the CDGS patient have increased reactivities with concanavalin A, demonstrating the presence of hybrid or oligomannose carbohydrate structures. However, bands 3 and 4.5 in HEMPAS erythrocytes have almost complete lack of poly(N-acetyllactosamine). Furthermore, CDGS type-II patients have a totally different clinical presentation and their erythrocytes do not show the serology typical of HEMPAS, suggesting that the genetic lesions responsible for these two diseases are possibly different.

Adolescent↗
PubMed1995

Characterization and modeling of membrane proteins using sequence analysis.

The current libraries of amino acid sequences of membrane proteins are a valuable resource for the analysis of elements common to these proteins. Multiple-sequence alignment techniques and the identification of conserved features of transmembrane segments have improved the prediction of membrane protein topology. Molecular modeling in combination with structural studies or site-directed mutagenesis is proving to be a powerful link between theory and experiment. Unfortunately, the number of high-resolution structures of intrinsic membrane proteins, although increased recently, presents a restricted and perhaps biased view of membrane protein structure.

Amino Acid Sequence↗
PubMed1995

Differential interaction of human renal P-glycoprotein with various metabolites and analogues of cyclosporin A.

Interactions of P-glycoprotein with several analogues and metabolites of cyclosporin A were studied to gain a better understanding of this immunosuppressant's mechanism of excretion and nephrotoxicity. Incorporation of [3H]azidopine into human renal P-glycoprotein in the presence of various concentrations of different cyclosporins was quantitated. Competitive [3H]azidopine photolabeling and 3H drug transport assays of CHRC5 multidrug-resistant cells were also conducted to evaluate effects of cyclosporins on P-glycoprotein function. Cyclosporins A [half-maximal inhibition constant (K0.5) = 20 nM] and G (K0.5 = 40 nM) blocked [3H]azidopine photolabeling of renal P-glycoprotein at very low concentrations, whereas higher concentrations of cyclosporin C (K0.5 = 500 nM) and metabolites 1, 17, and 21 (K0.5 = 200 nM) were required to inhibit photolabeling. Metabolites H and 8 were ineffective in inhibition of [3H]azidopine photolabeling of human renal P-glycoprotein. Similarly, cyclosporins A, C, and G were the best inhibitors of [3H]azidopine photolabeling of P-glycoprotein in multidrug-resistant C5 cells; the various metabolites were less effective. Cyclosporins A, C, and G also enhanced cellular accumulation of [3H]cyclosporin A and several other 3H-labeled compounds known to be transported by P-glycoprotein in multidrug-resistant C5 cells. Differential affinities of cyclosporin A metabolites for P-glycoprotein suggest considerable drug-binding site specificity. Our current hypothesis is that cyclosporin A may be more nephrotoxic than its metabolites by virtue of its superior ability to bind to and competitively inhibit urinary excretion of an endogenous P-glycoprotein substrate. Our findings provide the basis for future design and testing of new cyclosporin derivatives that have immunosuppressive activity yet may be less nephrotoxic because of their poor interaction with renal P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Identification of an internal topogenic signal sequence in human Band 3, the erythrocyte anion exchanger.

The insertion of Band 3, the human erythrocyte anion exchanger, into microsomal membranes was studied in an in vitro reticulocyte lysate translation system. Band 3 consists of a 43-kDa amino-terminal cytosolic domain and a carboxyl-terminal 52-kDa membrane domain containing up to 14 transmembrane segments with a single N-glycosylation site at Asn-642. Insertion of truncated Band 3 molecules into microsomal membranes was assayed by glycosylation, resistance to alkaline extraction, and tryptic removal of the cytosolic domain. Truncations containing either the first four or the last eight putative transmembrane segments were stably integrated into microsomes showing that an intact membrane domain was not required for membrane integration. Furthermore, the extracytosolic domain following the seventh transmembrane segment was properly translocated across the microsomal membrane and glycosylated whether the seventh transmembrane segment was the first, last, or the only transmembrane segment in the construct. The ability of the entire membrane domain, the truncated domain beginning with the seventh transmembrane segment, or the seventh transmembrane segment to insert into microsomes was dependent on the presence of the signal recognition particle receptor. The seventh transmembrane segment in Band 3 therefore has the topogenic properties of an internal signal sequence.

Amino Acid Sequence↗
PubMed1994

The Na+/H+ exchanger NHE-1 possesses N- and O-linked glycosylation restricted to the first N-terminal extracellular domain.

The ubiquitously-expressed human Na+H+ exchanger (NHE-1) contains three consensus sites (Asn-X-Ser/Thr) for N-linked glycosylation at asparagines 75, 370, and 410. The first extracellular loop is rich in serine and threonine residues which may contain O-linked carbohydrate. In order to determine unambiguously the sites of glycosylation and their role in biosynthesis and cation transport, site-directed mutagenesis at the individual potential N-glycosylation sites (Asn to Asp) was performed and all possible double and triple mutants were constructed. The mutated DNAs were expressed in PS120 hamster fibroblasts lacking endogenous exchanger, and the transfected cells were selected by their ability to survive acute intracellular acidification. All constructs produced functional exchangers that had transport rates and pharmacological profiles that were similar to that of wild-type. Immunoblot analysis of the expressed proteins with and without N-glycosidase F treatment showed that only the first N-glycosylation site (Asn 75) is utilized. In addition, treatment of NHE-1 with neuraminidase and O-glycosidase demonstrated that NHE-1 also contains O-linked oligosaccharide. Two forms of NHE-1 was consistently observed, a mature form with a molecular mass of 110,000 Da which contains N-linked and O-linked oligosaccharide and is expressed at the cell surface, and a lower molecular mass form (85,000 Da) present in the endoplasmic reticulum which only contains N-linked high-mannose oligosaccharide. NHE-3, an apically-expressed epithelial isoform which does not possess the N75 N-linked putative glycosylation site and any extracellular loops enriched in serine and threonine residues, does not exhibit any detectable glycosylation.

Amino Acid Sequence↗
PubMed1994

Asparagine-linked oligosaccharides are localized to single extracytosolic segments in multi-span membrane glycoproteins.

A comprehensive survey of mammalian multi-span (polytopic) membrane proteins showed that asparagine(N)-linked oligosaccharides are localized to single extracytosolic segments. In most membrane proteins this is because potential consensus sites for N-glycosylation (Asn-Xaa-Ser/Thr, X not equal to Pro) are not found in multiple extracytosolic segments. In functional proteins where consensus N-glycosylation sites are contained within more than one extracytosolic segment, only the first segment contains N-linked carbohydrate. An exception is the alpha-subunit of the Na+ channel, which consists of a duplicated structure containing two glycosylated segments. The average size of established N-glycosylated loops connecting two transmembrane segments is 62 residues, with the smallest glycosylated loop being 33 residues in size. N-glycosylated sites are more highly conserved than non-glycosylated (primarily cytosolic) sites and are more common toward the N-terminus of the membrane domain of multi-span membrane proteins. The optimal conditions for glycosylation of consensus sites within an extracytosolic domain of a multi-span membrane protein are (i) the acceptor site is well-spaced (greater than 10 residues) from the transmembrane domain, (ii) the loop is greater than 30 residues in size and (iii) the segment is the first in the protein to contain a suitable extracytosolic consensus site. The localization of N-linked oligosaccharide chains to a single protein segment suggests either glycosylation of multiple loops may compromise protein folding or function, or only a single polypeptide domain can be optimally glycosylated during biosynthesis in vivo.

Amino Acid Sequence↗
PubMed1994

Three-dimensional map of the dimeric membrane domain of the human erythrocyte anion exchanger, Band 3.

The electroneutral exchange of chloride and bicarbonate across the human erythrocyte membrane is facilitated by Band 3, a 911 amino acid glycoprotein consisting of a 43 kDa N-terminal cytosolic domain that binds the cytoskeleton, haemoglobin and glycolytic enzymes and a 52 kDa C-terminal membrane domain that mediates anion transport. Electron microscopy and three-dimensional image reconstruction of negatively stained two-dimensional crystals of the dimeric membrane domain revealed a U-shaped structure with dimensions of 60 x 110 A, and a thickness of 80 A. The structure is open on the top and at the sides, with the monomers in close contact at the base. The basal domain is 40 A thick and probably spans the lipid bilayer. The upper part of the dimer consists of two elongated protrusions measuring 25 x 80 A in projection, with a thickness of 40 A. The protrusions form the sides of a canyon, enclosing a wide space that narrows down and converges into a depression at the centre of the dimer on the top of the basal domain. This depression may represent the opening to a transport channel located at the dimer interface. Based on the available protein-chemical data, the two protrusions face the cytosolic side of the membrane and they appear to be dynamic.

Amino Acid Sequence↗
PubMed1994

Transmembrane aromatic amino acid distribution in P-glycoprotein. A functional role in broad substrate specificity.

Multidrug resistance (MDR) in cancer cells is associated with overexpression of P-glycoprotein (Pgp), a membrane protein which interacts with structurally diverse hydrophobic molecules of high membrane affinity. In an analysis of the molecular basis for this broad range of substrate specificity, we found that the transmembrane (TM) regions of Pgp are rich in highly conserved aromatic amino acid residues. Computer-generated three-dimensional model structures showed that a typical substrate, rhodamine 123, can intercalate between three to four phenylalanine side-chains in any of several Pgp TM helices with minimal protrusion of the drug into bulk lipid, and that five to six (of the 12 Pgp putative TM segments) helices can facilitate transport through creation of a sterically compatible pore. In contrast to the case for proteins involved in the transport of membrane-impermeable, relatively polar substrates, the "transport path" for Pgp substrates need not be polar, and may involve either an internal channel occupied largely by aromatic side-chains, or external gaps along TM helix-lipid interfaces. Weakly polar interactions between drug cationic sites and Pgp aromatic residues contribute additionally to overall protein/drug binding. The ability of Pgp to recognize and efflux structurally diverse molecules suggests that rather than a unique structure, the Pgp channel may maintain the intrinsic capacity to undergo wide-ranging drug-dependent dynamic reorganization.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Mammalian exchangers and co-transporters.

In the past year, novel mammalian exchanger and co-transporter isoforms have been characterized. Specialized subdomains within these oligomeric transporters have been shown to be involved in biosynthesis, targeting, transport and regulation. Progress on the structural front has been limited due to the lack of high-resolution structures, but transport mutants responsible for disease states continue to be identified.

Animals↗
PubMed1994

Interaction of rat kidney P-glycoprotein with a urinary component and various drugs including cyclosporin A.

The interaction of rat renal P-glycoprotein with various drugs and a hydrophobic component found in rat urine was studied to gain an understanding of both its transport function in kidney and its potential role in drug secretion and drug-induced nephrotoxicity. Rat kidney brush-border membranes (BBM) were photolabeled with [3H]azidopine, a calcium-channel blocker that covalently labeles P-glycoprotein. P-glycoprotein was immunoprecipitated with a rabbit polyclonal antibody against the human MDR1 protein (multidrug resistance gene class 1). The amount of [3H]azidopine incorporated into P-glycoprotein was quantitated following gel electrophoresis and fluorography. Photolabeling inhibition assays were conducted with a panel of drugs known to interact with P-glycoprotein in multidrug-resistant cells. Verapamil or quinidine [half-maximal inhibition constant (K0.5) = 1 microM], vinblastine (K0.5 = 3 microM), and doxorubicin or daunomycin (K0.5 = 10 microM) all blocked [3H]azidopine photolabeling of renal P-glycoprotein. Of the drugs tested, the immunosuppressant drug, cyclosporin A, interacted with kidney P-glycoprotein with the highest affinity (K0.5 = 50 nM). However, the cardiac glycoside, digoxin, failed to inhibit P-glycoprotein photolabeling. A hydrophobic rat urine extract prepared by reverse-phase chromatography also blocked photolabeling of renal P-glycoprotein. Our current hypothesis is that various drugs may inhibit urinary excretion of an endogenous substrate by virtue of their ability to bind with high affinity to P-glycoprotein. A hypothesis of drug-induced nephrotoxicity based on the interaction of various compounds like cyclosporin A with P-glycoprotein is presented.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Molecular characterization of the band 3 protein from Southeast Asian ovalocytes.

Southeast Asian ovalocytosis (SAO) is a hereditary form of elliptocytosis resulting in rigid, oval-shaped erythrocytes resistant to invasion by malaria parasites. The molecular defect is due to deletion of codons 400-408, encoding a 9-amino-acid sequence located at the boundary between the cytosol and the first transmembrane segment in Band 3, the erythrocyte anion transport protein. We have carried out an extensive characterization of Band 3 isolated from SAO erythrocytes which contain about 50% mutant Band 3. A slightly higher proportion of Band 3 in SAO erythrocytes was left associated with the cytoskeleton after extraction of ghost membranes with non-ionic detergents. Size exclusion high performance liquid chromatography analysis showed that SAO Band 3 contained a higher proportion of tetramers relative to dimers (50% tetramer) than normal Band 3 (33% tetramer). The circular dichroism spectrum of Band 3 from SAO erythrocytes was very similar to the spectrum for normal Band 3. Enzymatic deglycosylation and tomato lectin binding showed that SAO Band 3 lacked the polylactosaminyl oligosaccharide found on normal Band 3. SAO Band 3 was unable to bind the anion transport inhibitor 4-benzamido-4'-aminostilbene-2,2'-disulfonate, suggesting a dramatic alteration in the inhibitor binding site. In conclusion, deletion of 9 amino acids from Band 3 on the cytosolic side of the membrane affects the properties (glycosylation and inhibitor binding) of Band 3 on the opposite side of the membrane without dramatic changes in the secondary and quaternary structure of the protein.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1993

Non-random distribution of amino acids in the transmembrane segments of human type I single span membrane proteins.

The distribution of amino acids in the transmembrane segments and flanking regions of 115 human type I single span (amino terminus extracellular and carboxyl terminus cytosolic) plasma membrane proteins was found to be non-random. In this sample, Ile was preferentially localized to the amino-terminal region of the hydrophobic transmembrane segments, followed by Val, while Leu predominated in the carboxyl-terminal half of the segment. Although Gly residues were preferentially located in the transmembrane segment, this residue was excluded from the carboxyl-terminal and adjacent boundary regions. Aromatic residues (Tyr, Trp and Phe) occurred preferentially at the cytoplasmic boundary, with Trp also favored at the extracellular boundary. The extracellular flanking sequence amino-terminal to the transmembrane segment was enriched in residues predicted to initiate helix formation (Pro, Asn and Ser), while Arg and Lys were enriched in the cytoplasmic flank where they may function as topological determinants. The positional preferences of these particular amino acids within the transmembrane segment and flanking regions suggests that, in addition to lipid-protein interactions, these residues may participate in specific protein-protein interactions. A consensus sequence motif for type I membrane proteins is proposed and its role in the biosynthesis, folding, assembly and function of these segments is discussed.

Amino Acid Sequence↗
PubMed1993

Detergent interaction with band 3, a model polytopic membrane protein.

The interaction of band 3, the 95-kDa anion-exchange protein of the human erythrocyte membrane, with a variety of nonionic detergents was studied. Band 3 dimers (Stokes radius = 76 A) prepared in octaethylene glycol monododecyl ether (C12E8) could be exchanged into a variety of detergents by size-exclusion high-performance liquid chromatography (HPLC), with complete removal of C12E8 from band 3 being confirmed using radiolabeled detergent. Critical micellar concentration (cmc) values, determined for all detergents in the buffer used for HPLC analysis, ranged from 0.47 microM to 223 mM. Band 3 was found to aggregate in all detergents below their cmc, and concentrations of detergents 2-200 times the cmc were required to prevent aggregation. For detergents with a low cmc, it was important to ensure that the concentration of detergent micelles minimally equalled the concentration of protein. Hydrodynamic measurements and cross-linking studies showed that band 3 remained dimeric in most detergents above their cmc. Furthermore, circular dichroism and inhibitor binding studies supported the view that band 3 can retain its native structure after detergent exchange. Detergents with short alkyl chains (C8) denature band 3, while detergents with longer alkyl chains (C12) maintained the native structure of band 3. The ability to exchange band 3 into a variety of detergents with the maintenance of native structure is an essential prerequisite for crystallization trials. The results obtained in this study of band 3, a model polytopic (multispanning) membrane protein, may be generally applicable to other membrane proteins.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1993

Two-dimensional structure of the membrane domain of human band 3, the anion transport protein of the erythrocyte membrane.

The membrane domain of human erythrocyte Band 3 protein (M(r) 52,000) was reconstituted with lipids into two-dimensional crystals in the form of sheets or tubes. Crystalline sheets were monolayers with six-fold symmetry (layer group p6, a = b = 170 A, gamma = 60 degrees), whereas the symmetry of the tubular crystals was p2 (a = 104 A, b = 63 A, gamma = 104 degrees). Electron image analysis of negatively stained specimens yielded projection maps of the protein at 20 A resolution. Maps derived from both crystal forms show that the membrane domain is a dimer of two monomers related by two-fold symmetry, with each monomer consisting of three subdomains. In the dimer, two subdomains of each monomer form a roughly rectangular core (40 x 50 A in projection), surrounding a central depression. The third subdomain of the monomer measures approximately 15 x 25 A in projection and appears to be connected to the other two by a flexible link. We propose that the central depression may represent the channel for anion transport while the third subdomain appears not to be directly involved in channel formation.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1992

Interaction of P-glycoprotein with a hydrophobic component of rat urine.

The presence of an endogenous P-glycoprotein substrate in rat urine was examined by testing the ability of a hydrophobic extract to reverse multidrug resistance in CHO cells and to inhibit [3H]azidopine photolabelling. The accumulation of several hydrophobic drugs and dyes, known to be transported by P-glycoprotein, was dramatically enhanced in multidrug-resistant CHO cells (CHRC5) by a component contained in a hydrophobic extract prepared from rat urine by octadecyl (C18) reverse phase chromatography. The biological action of this urinary component involves a direct interaction with P-glycoprotein since it blocked photolabelling of the protein with [3H]azidopine. The effective concentration of the substance required to enhance drug accumulation and inhibit photolabelling was similar and within the range of its urinary content. These results suggest that a hydrophobic substance in urine may be an endogenous substrate of kidney P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗