Exofacial Loop Research Articles

The serological profile and molecular basis of a new partial D phenotype, DHR.

The Rh D antigen comprises a mosaic of at least 30 epitopes expressed on a 30-kD non-glycosylated Rh D polypeptide. The equivalent Rh CeEe polypeptide expressing the Rh C/c and E/e antigens differs in only 36 of the 417 amino acid residues. Partial D individuals have been described who fail to express a number of D epitopes. Serologic methods were applied with monoclonal anti-D to map epitopes on the red cells of a proposita aberrant D typing. Polymerase chain reaction (PCR) and DNA sequencing were also done. DNA sequence analysis derived by RT-PCR using total RNA isolated from peripheral blood of this person suggests two mechanisms for the genetic basis of this variants: one here gene conversion events result in the replacement of RHD gene exons with the equivalent RHCE exons; the second where point mutation in the RHD gene generates an amino acid substitution in the Rh D protein. We report here a new partial D, DHR, where a single point mutation (G to A at nucleotide 686) in exon 5 of the RHD gene results in a conservative amino acid substitution (Arg229Lys), in the predicted Rh D protein. This residue is localised on the fourth predicted exofacial loop of the Rh D polypeptide as determined by hydropathy analysis. This substitution results in the lack of epD 1, 2, 12 and 20 (30 epitope model) and indicates the involvement of loop 4, and in particular the requirement of Arg229, in the expression of these epitopes.

Complementation Studies with Co-expressed Fragments of the Human Red Cell Anion Transporter (Band 3; AE1): THE ROLE OF SOME EXOFACIAL LOOPS IN ANION TRANSPORT

We constructed cDNA clones encoding fragments of band 3 in which the membrane domain was truncated from either the N or the C terminus within each of the first four exofacial loops. The truncations containing the C terminus of the protein were fused with the cleavable N-terminal signal sequence of glycophorin A to facilitate the correct orientation of the most N-terminal band 3 membrane span. Cleavage of the glycophorin A signal sequence was observed, except when the truncation was in the first exofacial loop where the signal peptidase cleavage site was probably too close to the membrane. The anion transport activity of co-expressed complementary pairs of truncations which together contained the entire band 3 membrane domain was examined. The pairs of fragments divided in the third and fourth exofacial loops yielded transport activity, but the pair separated within the second exofacial loop was not active. We conclude that the integrity of the second exofacial loop, but not the third and fourth exofacial loops, is necessary for transport activity. The unusually stable association between the fragments divided in the second exofacial loop suggests that interactions may occur between polar surfaces on amphiphilic portions of the third and fifth transmembrane spans.

Proteolytic cleavage sites of native AE2 anion exchanger in gastric mucosal membranes.

The AE2 anion exchanger in pig and rabbit gastric mucosal membranes was subjected to limited proteolysis with trypsin, chymotrypsin, and papain, and to enzymatic N-deglycosylation. A monoclonal antibody to the AE2 C-terminal peptide was raised, characterized, and used to purify pig AE2 and its C-terminal cleavage products. Five distinct proteolytic cleavage sites within the AE2 transmembrane domain were defined by amino acid sequencing. The amino acid sequence of pig AE2 in the region encompassing the N-glycosylated Z-loop was also determined by RT-PCR. Tryptic cleavage of pig AE2 in the Z-loop produced C-terminal glycopeptides and was unaffected by deglycosylation, whereas the smaller rabbit AE2 C-terminal tryptic peptide lacked oligosaccharide, consistent with the respective amino acid sequences. The third consensus N-glycosylation site in pig Z-loop was heterogeneously glycosylated. Rapid papain cleavage in the Z-loop and slower cleavage in loop 7-8 produced C-terminal peptide products which were not N-glycosylated. Chymotryptic cleavage of the rabbit AE2 Z-loop required prior deglycosylation. Chymotryptic cleavage in the pig AE2 Z-loop produced C-terminal glycopeptides. Prior deglycosylation of pig AE2 unmasked novel, ionic strength-sensitive chymotryptic cleavage sites in the adjacent exofacial loop 7-8. These results provide experimental confirmation for some aspects of AE2 topography previously predicted from primary structure alone.

Molecular Cloning and Characterization of AqpZ, a Water Channel from Escherichia coli

The aquaporin family of molecular water channels is widely expressed throughout the plant and animal kingdoms. No bacterial aquaporins are known; however, sequence-related bacterial genes have been identified that encode glycerol facilitators (glpF). By homology cloning, a novel aquaporin-related DNA (aqpZ) was identified that contained no surface N-glycosylation consensus. The aqpZ RNA was not identified in mammalian mRNA by Northern analysis and exhibited bacterial codon usage preferences. Southern analysis failed to demonstrate aqpZ in mammalian genomic DNA, whereas a strongly reactive DNA was present in chromosomal DNA from Escherichia coli and other bacterial species and did not correspond to glpF. The aqpZ DNA isolated from E. coli contained a 693-base pair open reading frame encoding a polypeptide 28-38% identical to known aquaporins. When compared with other aquaporins, aqpZ encodes a 10-residue insert preceding exofacial loop C, truncated NH2 and COOH termini, and no cysteines at known mercury-sensitive sites. Expression of aqpZ cRNA conferred Xenopus oocytes with a 15-fold increase in osmotic water permeability, which was maximal after 5 days of expression, was not inhibited with HgCl2, exhibited a low activation energy (Ea = 3.8 kcal/mol), and failed to transport nonionic solutes such as urea and glycerol. In contrast, oocytes expressing glpF transported glycerol but exhibited limited osmotic water permeability. Phylogenetic comparison of aquaporins and homologs revealed a large separation between aqpZ and glpF, consistent with an ancient gene divergence.

Ca(2+)- and GTP[gamma S]-induced translocation of the glucose transporter, GLUT-4, to the plasma membrane of permeabilized cardiomyocytes determined using a novel immunoprecipitation method.

In cardiomyocytes glucose transport is activated not only by insulin but also by contractile activity that causes translocation of the glucose transporter, GLUT-4, from intracellular vesicles to the plasma membrane. The latter effect may possibly be mediated by intracellular Ca2+, as suggested by previous studies. To investigate the role of Ca2+, we permeabilized neonatal rat myocytes with alpha-toxin and incubated them for 1 h either at a pCa (i.e.--log10 [Ca2+]) of 8 (control) or at a pCa of 5 in the presence of adenosine 5'-triphosphate (ATP). Translocation of GLUT-4 was then monitored by a novel immunoprecipitation method using a peptide antibody directed against an exofacial (extracellular) loop of GLUT-4 (residues 58-80). Incorporation of GLUT-4 into the plasmalemma was stimulated 1.8-fold by 10 microM Ca2+ and 1.7-fold by insulin (as in the case of intact cells). The insulin effect was Ca2+ independent, i.e. it was identical in the absence and presence of Ca2+ (10 microM). Guanosine 5'-O-(3-thio-triphosphate) (GTP[gamma S]), which was inactive in intact cells, also caused translocation of GLUT-4 in permeabilized cardiomyocytes. Thus, incorporation of GLUT-4 into the plasma membrane was enhanced 2.5-fold by 200 microM GTP[gamma S] in the virtual absence of Ca2+ (pCa 8) and even 3.5-fold at 10 microM free Ca2+. We conclude that an increase in intracellular Ca2+ concentration increases GLUT-4 translocation of (permeabilized) cardiomyocytes to a similar extent as do insulin and GTP[gamma S] in the absence of Ca2+, but that the effects of Ca2+ and GTP[gamma S] may be additive.

Immune responses to band 3 neoantigens on Plasmodium falciparum-infected erythrocytes in subjects living in an area of intense malaria transmission are associated with low parasite density and high hematocrit value.

During the intracellular development of the human malarial parasite, Plasmodium falciparum, cryptic regions of the erythrocyte band 3 protein are exposed. Antibodies against these band 3-related neoantigens block cytoadherence, and peptides based on amino acid sequences of putative exofacial loops of band 3 protein block the in vitro and in vivo adherence of P. falciparum-infected erythrocytes. At present, it is not known whether reactivity to these antigens is related to exposure to the malaria parasite or is correlated with protective immunity. The reactivities of plasma to peptides containing amino acid sequences of putative exofacial loops 3 and 7 of human band 3 protein were determined for children and adults living in an area of perennial malaria transmission (Liberia) and for donors who had never been exposed to malaria (Denmark). Plasma samples from children and adults living in an area of intense malaria transmission showed a much higher reactivity with the band 3 peptides than did those from nonimmune individuals. High reactivity to the loop 3 peptide (amino acids 546 to 555) was correlated with lower mean parasite density in children in the 5- to 9-year-old age group. The presence of antibodies against loop 3 and 7 peptides was not associated with a low packed erythrocyte volume (hematocrit); in fact, higher-than-average reactivities to both peptides were positively correlated with high hematocrit values, indicating that antibodies which specifically recognize the band 3-related neoantigens are not involved in hemolysis (autoimmunity).

Topology of the Glut 1 glucose transporter deduced from glycosylation scanning mutagenesis.

The erythrocyte glucose transporter (Glut 1) is predicted to contain 12 membrane-spanning domains based on the hydropathy plot of its deduced amino acid sequence. The membrane topology of Glut 1 was analyzed by a scanning mutagenesis procedure in which the glycosylated exofacial domain of Glut 4 was inserted independently into each of the putative hydrophilic soluble domains of an aglyco-Glut 1 construct. The transporter mutants were expressed both in vitro using a rabbit reticulocyte lysate translation system and in vivo in Xenopus oocytes. The cytoplasmic or exofacial orientation of each soluble domain was inferred from the glycosylation state of the corresponding insertion mutant. The results from the cell-free system were aberrant in that two topological orientations were observed when the epitope was inserted into any of the short cytoplasmic loops or the NH2 terminus. The in vivo data, however, were in complete agreement with the proposed 12-helix model. Therefore, the multiple topologies observed in vitro probably resulted from the inability of the cell-free system to facilitate the proper folding of the insertion mutants into the membrane. 2-Deoxyglucose uptake data on the glycosylation mutants indicated that epitope insertion into the NH2 terminus, the large central loop, or the second, third, or fifth exofacial loop had no dramatic effect on the activity of the transporter. However, insertion into the other soluble domains either completely abolished or significantly reduced transport activity.

Tyrosine kinase-deficient mutant human insulin receptors (Met1153-->Ile) overexpressed in transfected rat adipose cells fail to mediate translocation of epitope-tagged GLUT4.

Insulin regulates essential pathways for growth, differentiation, and metabolism in vivo. We report a physiologically relevant system for dissecting the molecular mechanisms of insulin signal transduction related to glucose transport. This is an extension of our recently reported method for transfection of DNA into rat adipose cells in primary culture. In the present work, cDNA coding for GLUT4 with an epitope tag (HA1) in the first exofacial loop is used as a reporter gene so that GLUT4 translocation can be studied exclusively in transfected cells. Insulin stimulates a 4.3-fold recruitment of transfected epitope-tagged GLUT4 to the cell surface. Cells cotransfected with the reporter gene and the human insulin receptor gene show an increase in cell surface GLUT4 in the basal state (no insulin) to levels comparable to those seen with maximal insulin stimulation of cells transfected with the reporter gene alone. In contrast, cells overexpressing a naturally occurring tyrosine kinase-deficient mutant insulin receptor (Met1153-->Ile) show no increase in the basal cell surface GLUT4 and no shift in the insulin dose-response curve relative to cells transfected with the reporter gene alone. These results demonstrate that insulin receptor tyrosine kinase activity is essential in insulin-stimulated glucose transport in adipose cells.

Insulin action on the internalization of the GLUT4 glucose transporter in isolated rat adipocytes

A novel method was developed to measure relative amounts of the GLUT4 glucose transporter on the surface of intact fat cells and to monitor the action of insulin on cell surface glucose transporters as they internalize into intracellular membranes. The method takes advantage of two predicted trypsin cleavage sites in the major exofacial loop of this transporter protein. Treatment of cyanide-poisoned rat adipocytes with 1 mg/ml trypsin at 37 degrees C for 30 min produced an immunoreactive GLUT4 protein species in subsequently isolated plasma membranes that migrated with higher mobility (apparent M(r) = 35,000) than native GLUT4 (apparent M(r) = 46,000) on SDS-polyacrylamide gel electrophoresis. This proteolyzed GLUT4 protein was absent in the intracellular low density microsomes. Insulin treatment of adipocytes for 20 min prior to sequential additions of cyanide and trypsin caused a 16-fold increase in the proteolytically cleaved GLUT4 species. Incubation of fresh fat cells with trypsin caused a rapid and progressive appearance of the proteolyzed GLUT4 species in the intracellular low density membranes as well as plasma membranes. After 5 min of trypsinization, 66% of the total cleaved GLUT4 in these cells had moved into the low density membranes. Insulin treatment markedly decreased the internalized cleaved GLUT4 to 20% of the total. These data indicate the following: 1) trypsinization of the GLUT4 transporter protein on intact fat cells is a convenient means to monitor the extent of transporter recruitment to the plasma membrane by insulin, as well as to estimate GLUT4 internalization rates; and 2) the action of insulin on glucose transporter redistribution to the cell surface is associated with a marked inhibition of the fraction of cell surface GLUT4 transporters internalized per unit time.

Expression of mRNA of beta 1- and beta 2-adrenergic receptors in Xenopus oocytes results from structurally distinct receptor mRNAs.

Poly(A)+-selected RNA prepared from cells or tissues that express a homogeneous population of either beta 1- or beta 2-adrenergic receptors was isolated and then microinjected into Xenopus laevis oocytes. Following microinjection, the expression of beta-adrenergic receptors was assessed by equilibrium radioligand binding analysis using the antagonist ligand [3H]dihydroalprenolol. The pharmacology of the newly- expressed beta-adrenergic receptors in oocyte membranes was the same as that of the original tissue used as a source of RNA. Hybridization of nick-translated cDNA of hamster beta 2-adrenergic receptor to poly(A)+-selected RNA from tissues containing beta 2-adrenergic receptors was to a mRNA species of 2.2 kilobases. In contrast, hybridization of the cDNA probe to poly(A)+-selected RNA from tissues containing beta 1-adrenergic receptors was to a mRNA species of 2.0 kilobases. A single-stranded fragment of hamster beta 2-adrenergic receptor cDNA corresponding to nucleotides 730-886 was isolated and uniformly radiolabeled. This region of the gene is predicted to encode for the entire second exofacial loop (L4-5), the entire fifth transmembrane-spanning region, and the first 5 amino acid residues of the third cytoplasmic loop (L5-6) of the beta 2-adrenergic receptor. Hybridization at 48 and 56 degrees C of poly(A)+-selected RNA prepared from sources that express either beta 1 or beta 2-adrenergic receptors to the antisense orientation strand of this region of the beta 2-adrenergic receptor cDNA was followed by S1 endonuclease digestion of nonhybridized sequences. At 48 degrees C, S1-resistant hybrids from both sources of RNA protected the probe from S1 endonuclease digestion. At 56 degrees C, however, only the RNA prepared from the source of beta 2-adrenergic receptors protected the probe from S1 endonuclease digestion. These results demonstrate that the mRNAs encoding for the structurally homologous beta 1- and beta 2-adrenergic receptors are distinct in the pharmacological specificity of their translation products and in their size and structure.