Glycoprotein hormone GalNAc-4-sulphotransferase.

The glycoprotein hormones lutropin (LH) and thyrotropin and a limited number of additional glycoproteins bear carbohydrate structures terminating with the unique sequence SO(4)-4-GalNAcbeta1,4GlcNAcbeta that has been conserved in the glycoprotein hormones of all vertebrate species. Synthesis of these structures is mediated by a protein-specific beta1,4GalNAc-transferase and a GalNAc-4-sulphotransferase (GalNAc-4-ST1). GalNAc-4-ST1 is a member of a family of sulphotransferases that includes HNK-1 sulphotransferase, chondroitin-4-sulphotransferases-1-3 and dermatan-4-sulphotransferase-1. With the exception of HNK-1-ST, these sulphotransferases add sulphate to the C-4 hydroxy group of either terminal or non-terminal beta1,4-linked GalNAc. GalNAc-4-ST1 is most highly expressed in pituitary, cerebellum and other regions of the brain. The terminal GalNAcSO(4) on LH is recognized by the cysteine-rich domain of the mannose/GalNAc-4-SO(4) receptor located in hepatic endothelial cells. Each cysteine-rich domain binds a single terminal GalNAc-4-SO(4), and the receptor must form non-covalently associated homodimers in order to simultaneously engage two GalNAc-4-SO(4) moieties on separate oligosaccharides with sufficient affinity to form stable complexes. The receptor mediates the clearance of LH from the blood. This clearance, in conjunction with the stimulated release of hormone from dense core granules in pituitary gonadotroph cells, is required to produce the episodic rise and fall in LH levels needed for optimal oestrogen production during the implantation of embryos in the uterus.

Molecular cloning and characterization of a dermatan-specific N-acetylgalactosamine 4-O-sulfotransferase.

We have identified and characterized an N-acetylgalactosamine-4-O-sulfotransferase designated dermatan-4-sulfotransferase-1 (D4ST-1) (GenBank(TM) accession number AF401222) based on its homology to HNK-1 sulfotransferase. The cDNA predicts an open reading frame encoding a type II membrane protein of 376 amino acids with a 43-amino acid cytoplasmic domain and a 316-amino acid luminal domain containing two potential N-linked glycosylation sites. D4ST-1 has significant amino acid identity with HNK-1 sulfotransferase (21.4%), N-acetylgalactosamine-4-O-sulfotransferase 1 (GalNAc-4-ST1) (24.7%), N-acetylgalactosamine-4-O-sulfotransferase 2 (GalNAc-4-ST2) (21.0%), chondroitin-4-O-sulfotransferase 1 (27.3%), and chondroitin-4-O-sulfotransferase 2 (22.8%). D4ST-1 transfers sulfate to the C-4 hydroxyl of beta1,4-linked GalNAc that is substituted with an alpha-linked iduronic acid (IdoUA) at the C-3 hydroxyl. D4ST-1 shows a strong preference in vitro for sulfate transfer to IdoUAalpha1,3GalNAcbeta1,4 that is flanked by GlcUAbeta1,3GalNAcbeta1,4 as compared with IdoUAalpha1,3GalNAcbeta1,4 flanked by IdoUAalpha1,3GalNAcbeta1,4. The specificity of D4ST-1 when assayed in vitro suggests that the addition of sulfate to GalNAc occurs immediately after epimerization of GlcUA to IdoUA. The open reading frame of D4ST-1 is encoded by a single exon located on human chromosome 15q14. Northern blot analysis reveals a single 2.4-kilobase transcript. D4ST-1 message is expressed in virtually all tissues at some level but is most highly expressed in pituitary, placenta, uterus, and thyroid. The properties of D4ST-1 indicate that sulfation of the GalNAc moieties in dermatan is mediated by a distinct GalNAc-4-O-sulfotransferase and occurs following epimerization of GlcUA to IdoUA.

Structure of the pore-forming transmembrane domain of a ligand-gated ion channel.

The structure of the pore-forming transmembrane domain of the nicotinic acetylcholine receptor from Torpedo has been investigated by infrared spectroscopy. Treatment of affinity-purified receptor with either Pronase or proteinase K digests the extramembranous domains (roughly 75% of the protein mass), leaving hydrophobic membrane-imbedded peptides 3-6 kDa in size that are resistant to peptide (1)H/(2)H exchange. Infrared spectra of the transmembrane domain preparations exhibit relatively sharp and symmetric amide I and amide II band contours centered near 1655 and 1545 cm(-)1, respectively, in both (1)H(2)O and (2)H(2)O. The amide I band is very similar to the amide I bands observed in the spectra of alpha-helical proteins, such as myoglobin and bacteriorhodopsin, that lack beta structure and exhibit much less beta-sheet character than is observed in proteins with as little as 20% beta sheet. Curve-fitting estimates 75-80% alpha-helical character, with the remaining peptides likely adopting extended and/or turn structures at the bilayer surface. Infrared dichroism spectra are consistent with transmembrane alpha-helices oriented perpendicular to the bilayer surface. The evidence strongly suggests that the transmembrane domain of the nicotinic receptor, the most intensively studied ligand-gated ion channel, is composed of five bundles of four transmembrane alpha-helices.

The mannose/N-acetylgalactosamine-4-SO4 receptor displays greater specificity for multivalent than monovalent ligands.

Recognition of carbohydrates on glycosylated molecules typically requires multivalent interactions with receptors. Monovalent forms of terminal saccharides engaged by the receptor binding sites typically display weak affinities in the mm range and poor specificity. In contrast, multivalent forms of the same saccharides are bound with strong affinity (10(-7)-10(-9) m) and significantly greater specificity. Although multivalency can readily account for increased affinity, the molecular basis for enhanced specificity is not well understood. We have examined the specificity of the cysteine-rich domain of the mannose/GalNAc-4-SO4 receptor using monovalent and multivalent forms of the trisaccharide GalNAcbeta1,4GlcNAcbeta1,2Manalpha (GGnM) sulfated at either the C4 (S4GGnM) or C3 (S3GGnM) hydroxyl of the terminal GalNAc. Monovalent S4GGnM and S3GGnM have K(i) values of 25.8 and 16.2 microm, respectively. Multivalent conjugates of the same GalNAc-4-SO4- and GalNAc-3-SO4-bearing trisaccharides (6.7 mol of trisaccharide/mol of bovine serum albumin) have K(i) values of 0.013 and 0.170 microm, respectively. The 2000-fold versus 95-fold change in affinity seen for the multivalent forms of these 4-sulfated and 3-sulfated trisaccharides reflects a difference in the impact of conformational entropy. A large fraction of the SO4-3-GalNAc structures exists in a form that is not favorable for binding to the Cys-rich domain. This reduces the effective concentration of SO4-3-GalNAc as compared with SO4-4-GalNAc under the same conditions and results in a markedly lower association rate. This difference in association rate accounts for the 12-fold difference in the rate of clearance from the blood seen with S4GGnM-BSA and S3GGnM-BSA in vivo.

Molecular cloning and expression of an N-acetylgalactosamine-4-O-sulfotransferase that transfers sulfate to terminal and non-terminal beta 1,4-linked N-acetylgalactosamine.

We have identified and characterized an N-acetylgalactosamine-4-O-sulfotransferase designated GalNAc-4-ST2 (GenBank(TM) accession number ) based on its homology to HNK-1 sulfotransferase (HNK-1 ST). The cDNA predicts an open reading frame encoding a type II membrane protein of 443 amino acids with a 12-amino acid cytoplasmic domain, a 23-amino acid transmembrane domain, and a 408-amino acid luminal domain containing four potential N-linked glycosylation sites. GalNAc-4-ST2 displays a high degree of amino acid sequence identity with GalNAc-4-ST1 (46%), HNK-1 ST (23%), chondroitin 4-O-sulfotransferase-1 (C4ST-1) (27%), and chondroitin 4-O-sulfotransferase-2 (C4ST-2) (24%). GalNAc-4-ST2 transfers sulfate to the C-4 hydroxyl of terminal beta1,4-linked GalNAc in the sequence GalNAc-beta1,4GlcNAcbeta-R found on N-linked oligosaccharides and nonterminal beta1,4-linked GalNAc in chondroitin and dermatan. The translated region of GalNAc-4-ST2 is encoded by five exons located on human chromosome 18q11.2. Northern blot analysis reveals a 2.1-kilobase transcript. GalNAc-4-ST2 message is most highly expressed in trachea and to a lesser extent in heart, liver, pancreas, salivary gland, and testis. The I.M.A.G.E. cDNA clone 49547 contains a putative GalNAc-4-ST2 splice form with an open reading frame encoding a protein of 358 amino acids that lacks the transmembrane domain and the stem region. This form of GalNAc-4-ST2 is not retained by transfected cells and is active against chondroitin but not terminal beta1,4-linked GalNAc. Thus, as with GalNAc-4-ST1, sequences N-terminal to the catalytic domain contribute to the specificity of GalNAc-4-ST2 toward terminal beta1,4-linked GalNAc.

A conformational intermediate between the resting and desensitized states of the nicotinic acetylcholine receptor.

The structural changes induced in the nicotinic acetylcholine receptor by two noncompetitive channel blockers, proadifen and phencyclidine, have been studied by infrared difference spectroscopy and using the conformationally sensitive photoreactive noncompetitive antagonist 3-(trifluoromethyl)-3-m-([(125)I]iodophenyl)diazirine. Simultaneous binding of proadifen to both the ion channel pore and neurotransmitter sites leads to the loss of positive markers near 1663, 1655, 1547, 1430, and 1059 cm(-)(1) in carbamylcholine difference spectra, suggesting the stabilization of a desensitized conformation. In contrast, only the positive markers near 1663 and 1059 cm(-)(1) are maximally affected by the binding of either blocker to the ion channel pore suggesting that the conformationally sensitive residues vibrating at these two frequencies are stabilized in a desensitized-like conformation, whereas those vibrating near 1655 and 1430 cm(-)(1) remain in a resting-like state. The vibrations at 1547 cm(-)(1) are coupled to those at both 1663 and 1655 cm(-)(1) and thus exhibit an intermediate pattern of band intensity change. The formation of a structural intermediate between the resting and desensitized states in the presence of phencyclidine is further supported by the pattern of 3-(trifluoromethyl)-3-m-([(125)I]iodophenyl)diazirine photoincorporation. In the presence of phencyclidine, the subunit labeling pattern is distinct from that observed in either the resting or desensitized conformations; specifically, there is a concentration-dependent increase in the extent of photoincorporation into the delta-subunit. Our data show that domains of the nicotinic acetylcholine receptor interconvert between the resting and desensitized states independently of each other and suggest a revised model of channel blocker action that involves both low and high affinity agonist binding conformational intermediates.

Urine GABA levels in ovarian cancer patients: elevated GABA in malignancy.

The association of malignancy with elevated diamine oxidase (DAO), an enzyme producing gamma-aminobutyric acid (GABA) is well documented. In ovarian cancer, increased DAO occurs in the malignant tissues and plasma. Since higher DAO levels cause GABA accumulation, elevated GABA may occur in ovarian cancer and be reflected in urine. We tested this hypothesis and found that half the ovarian cancer patients had a clearly elevated urine GABA, a result that is in agreement with previous reports on DAO and malignancy. The published findings on DAO, GABA and malignancy suggest that elevated GABA is associated with cancer. This proposal could lead to a GABA urine monitor or new directions in cancer treatment or research.

Molecular cloning and expression of the pituitary glycoprotein hormone N-acetylgalactosamine-4-O-sulfotransferase.

N-Linked oligosaccharides terminating with the sequence SO(4)-4-GalNAcbeta1,4GlcNAcbeta1,2Manalpha are present on the pituitary hormones lutropin (LH), thyrotropin, and pro-opiomelanocortin. The sulfated structures on LH are essential for expression of its biologic function in vivo. We have cloned the N-acetylgalactosamine-4-sulfotransferase (GalNAc-4-ST1, GenBank(TM) accession number ), which mediates sulfate addition to the N-linked oligosaccharides on LH and other pituitary glycoproteins with terminal (beta1,4-linked GalNAc based on its homology to HNK-1 sulfotransferase (HNK-1 ST). GalNAc-4-ST1 displays 23% identity to HNK-1 ST and 28% to chondroitin 4-sulfotransferase 1 (C4ST-1) and 26% to chondroitin 4-sulfotransferase 2 (C4ST-2). The cDNA predicts a type II transmembrane protein of 424 amino acids with four potential N-linked glycosylation sites and a single membrane-spanning domain. GalNAc-4-ST1 has putative 5'-phosphosulfonate and 3'-phosphate binding sites. Three more carboxyl-terminal regions of unknown function also show a high degree of identity with HNK-1 ST, C4ST-1, and C4ST-2. The membrane-bound form of GalNAc-4-ST1 transfers sulfate to GalNAcbeta1, 4GlcNAcbeta-R but not to chondroitin, whereas truncated forms of GalNAc-4-ST1 that are released into the medium transfer sulfate to both GalNAcbeta1,4GlcNAcbeta-R and chondroitin. The first 118 amino acids of GalNAc-4-ST1 appear to contribute to both its activity and specificity for terminal beta1,4-linked GalNAc. GalNAc-4-ST1 also efficiently transfers sulfate to N-linked oligosaccharides on native LH and other glycoproteins terminating with beta1,4-linked GalNAc. A single transcript of 2.4 kilobases is most highly expressed in the pituitary and other regions of the central nervous system. The GalNAc-4-ST1 gene is located on human chromosome 19q13.1.

Molecular basis of lutropin recognition by the mannose/GalNAc-4-SO4 receptor.

The circulatory half-life of the glycoprotein hormone lutropin (LH) is precisely regulated by the mannose (Man)/GalNAc-4-SO(4) receptor expressed in hepatic endothelial cells. Rapid clearance from the circulation contributes to the episodic rise and fall of LH levels that is essential for maximal stimulation of the G protein-coupled LH receptor. We have defined two molecular forms of the Man/GalNAc-4-SO(4) receptor that differ in ligand specificity, cell and tissue expression, and function. The form expressed by hepatic endothelial cells binds GalNAc-4-SO(4)-bearing ligands and regulates hormone circulatory half-life, whereas the form expressed by macrophages binds Man-bearing ligands and may play a role in innate immunity. We demonstrate that the GalNAc-4-SO(4)-specific form in hepatic endothelial cells is dimeric whereas the Man-specific form in lung macrophages is monomeric, accounting for the different ligand specificities of the receptor expressed in these tissues. Two cysteine-rich domains, each of which binds a single GalNAc-4-SO(4), are required to form stable complexes with LH. The kinetics of LH binding by the GalNAc-4-SO(4)-specific form of the receptor in conjunction with its rate of internalization from the cell surface make it likely that only two of the four terminal GalNAc-4-SO(4) moieties present on native LH are engaged before receptor internalization. As a result, the rate of hormone clearance will remain constant over a wide range of LH concentrations and will not be sensitive to variations in the number of terminal GalNAc-4-SO(4) moieties as long as two or more are present on multiple oligosaccharides.

Expression cloning of a new member of the ABO blood group glycosyltransferases, iGb3 synthase, that directs the synthesis of isoglobo-glycosphingolipids.

The large array of different glycolipids described in mammalian tissues is a reflection, in part, of diverse glycosyltransferase expression. Herein, we describe the cloning of a UDP-galactose: beta-d-galactosyl-1,4-glucosylceramide alpha-1, 3-galactosyltransferase (iGb(3) synthase) from a rat placental cDNA expression library. iGb(3) synthase acts on lactosylceramide, LacCer (Galbeta1,4Glcbeta1Cer) to form iGb(3) (Galalpha1,3Galbeta1, 4Glcbeta1Cer) initiating the synthesis of the isoglobo-series of glycosphingolipids. The isolated cDNA encoded a predicted protein of 339 amino acids, which shows extensive homology (40-50% identity) to members of the ABO gene family that includes: murine alpha1, 3-galactosyltransferase, Forssman (Gb(5)) synthase, and the ABO glycosyltransferases. In contrast to the murine alpha1, 3-galactosyltransferase, iGb(3) synthase preferentially modifies glycolipids over glycoprotein substrates. Reverse transcriptase-polymerase chain reaction revealed a widespread tissue distribution of iGb(3) synthase RNA expression, with high levels observed in spleen, thymus, and skeletal muscle. As an indirect consequence of the expression cloning strategy used, we have been able to identify several potential glycolipid biosynthetic pathways where iGb(3) functions, including the globo- and isoglobo-series of glycolipids.

Cloning of Gb3 synthase, the key enzyme in globo-series glycosphingolipid synthesis, predicts a family of alpha 1, 4-glycosyltransferases conserved in plants, insects, and mammals.

We have cloned Gb(3) synthase, the key alpha1, 4-galactosyltransferase in globo-series glycosphingolipid (GSL) synthesis, via a phenotypic screen, which previously yielded iGb(3) synthase, the alpha1,3-galactosyltransferase required in isoglobo-series GSL (Keusch, J. J., Manzella, S. M., Nyame, K. A., Cummings, R. D., and Baenziger, J. U. (2000) J. Biol. Chem. 33). Both transferases act on lactosylceramide, Galbeta1,4Glcbeta1Cer (LacCer), to produce Gb(3) (Galalpha1,4LacCer) or iGb(3) (Galalpha1, 3LacCer), respectively. GalNAc can be added sequentially to either Gb(3) or iGb(3) yielding globoside and Forssman from Gb(3), and isogloboside and isoForssman from iGb(3). Gb(3) synthase is not homologous to iGb(3) synthase but shows 43% identity to a human alpha1,4GlcNAc transferase that transfers a UDP-sugar in an alpha1, 4-linkage to a beta-linked Gal found in mucin. Extensive homology (35% identity) is also present between Gb(3) synthase and genes in Drosophila melanogaster and Arabidopsis thaliana, supporting conserved expression of an alpha1,4-glycosyltransferase, possibly Gb(3) synthase, throughout evolution. The isolated Gb(3) synthase cDNA encodes a type II transmembrane glycosyltransferase of 360 amino acids. The highest tissue expression of Gb(3) synthase RNA is found in the kidney, mesenteric lymph node, spleen, and brain. Gb(3) glycolipid, also called P(k) antigen or CD77, is a known receptor for verotoxins. CHO cells that do not express Gb(3) and are resistant to verotoxin become susceptible to the toxin following transfection with Gb(3) synthase cDNA.

Priming of strong, broad, and long-lived HIV type 1 p55gag-specific CD8+ cytotoxic T cells after administration of a virus-like particle vaccine in rhesus macaques.

Despite advances in the clinical management of HIV infection, using combinations of antiretroviral pharmaceuticals, a safe and efficacious vaccine is needed to limit the AIDS pandemic. It is now thought that an effective HIV-1 vaccine should prime both cross-neutralizing antibodies and long-lasting cytotoxic CD8+ T lymphocytes (CTLs) recognizing multiple codominant HIV-1 epitopes. To that end, many novel vaccine strategies have been tested. However, only a few of these strategies, beside those relying on live-attenuated viruses, are able to prime strong CTL responses in nonhuman primates and humans. In this study, three rhesus macaques were immunized with HIV-1 p55gag virus-like particles (VLPs) in the absence of adjuvant to assess the potential of such a vaccine to prime CTL responses. After intramuscular injection of p55gag VLP, all three animals mounted CTL responses against HIV-1 p55gag. Notably, these CTLs primed by vaccination recognized naturally processed peptides and were long lived (>8.5 months) both in the peripheral blood and draining lymph node. Furthermore, these CTLs were directed against multiple HIV-1 p55gag epitopes. This indicated that immunization with p55gag VLP primes strong MHC class I-restricted, CD8+ cell-mediated immune responses and suggested that HIV-1 p55gag VLPs should be a reasonable vaccine candidate, when combined with strategies priming cross-neutralizing antibodies.

Effect of membrane lipid composition on the conformational equilibria of the nicotinic acetylcholine receptor.

The effects of cholesterol (Chol) and an anionic lipid, dioleoylphosphatidic acid (DOPA) on the conformational equilibria of the nicotinic acetylcholine receptor (nAChR) have been investigated using Fourier transform infrared difference spectroscopy. The difference between spectra recorded in the presence and absence of agonist from the nAChR reconstituted into 3:1:1 egg phosphatidylcholine (EPC)/DOPA/Chol membranes exhibits positive and negative bands that serve as markers of the structural changes associated with the resting to desensitized conformational change. These markers are absent in similar difference spectra recorded from the nAChR reconstituted into EPC membranes lacking both Chol and DOPA, indicating that the nAChR cannot undergo conformational change in response to agonist binding. When low levels of either Chol or DOPA up to 25 mol % of the total lipid are included in the EPC membranes, the markers suggest the predominant stabilization of a conformation that is a structural intermediate between the resting and desensitized states. At higher levels of either Chol or DOPA, the nAChR is stabilized in a conformation that is capable of undergoing agonist-induced desensitization, although DOPA appears to be required for the nAChR to adopt a conformation fully equivalent to that found in native and 3:1:1 EPC/DOPA/Chol membranes. The ability of these two structurally diverse lipids, as well as others (Ryan, S. E., Demers, C. N., Chew, J. P., Baenziger, J. E. (1996) J. Biol. Chem. 271, 24590-24597), to modulate the functional state of the nAChR suggests that lipids act on the nAChR via an indirect effect on some physical property of the lipid bilayer. The data also suggest that anionic lipids are essential to stabilize a fully functional nAChR. We propose that membrane fluidity modulates the relative populations of nAChRs in the resting and desensitized states but that subtle structural changes in the presence of anionic lipids are essential for full activity.

Internal dynamics of the nicotinic acetylcholine receptor in reconstituted membranes.

The structure and 1H/2H exchange kinetics of affinity-purified nAChR reconstituted into egg phosphatidylcholine membranes with increasing levels of either dioleoylphosphatidic acid (DOPA) or cholesterol (Chol) have been examined using infrared spectroscopy. All spectra of the reconstituted nAChR membranes recorded after 72 h in 2H2O exhibit comparable amide I band shapes, suggesting a similar secondary structure for the nAChR in each lipid environment. Increasing levels of either DOPA or Chol, however, lead to an increasing intensity of the amide II band, indicating a decreasing proportion of nAChR peptide hydrogens that have exchanged for deuterium. Spectra recorded as a function of time after exposure of the nAChR to 2H2O show that the presence of either lipid slows down the 1H/2H exchange of those peptide hydrogens that normally exchange on the minutes to hours time scale. The slowing of peptide 1H/2H exchange correlates with both an increasing ability of the nAChR to undergo agonist-induced conformational change [Baenziger, J. E., Morris, M.-L., Darsaut, T. E., and Ryan, S. E. (1999) in preparation] and possibly a decreasing membrane fluidity. Our data suggest that lipid composition dependent changes in nAChR peptide 1H/2H exchange kinetics reflect altered internal dynamics of the nAChR. Lipids may influence protein function by changing the internal dynamics of integral membrane proteins.

A structure-based approach to nicotinic receptor pharmacology.

Infrared difference spectroscopy has been used to examine the structural effects of local anesthetic (LA) binding to the nicotinic acetylcholine receptor (nAChR). Several LAs induce subtle changes in the vibrational spectrum of the nAChR over a range of concentrations consistent with their reported nAChR-binding affinities. At concentrations of the desensitizing LAs prilocaine and lidocaine consistent with their binding to the ion channel pore, the vibrational changes suggest the stabilization of an intermediate conformation that shares structural features in common with both the resting and desensitized states. Higher concentrations of prilocaine and lidocaine, as well as the LA dibucaine, lead to additional binding to the neurotransmitter-binding site, the formation of physical interactions (most notably cation-tyrosine interactions) between LAs and neurotransmitter-binding-site residues, and the subsequent formation of a presumed desensitized nAChR. Although concentrations of the LA tetracaine consistent with binding to the ion channel pore elicit a reversed pattern of spectral changes suggestive of a resting state-like nAChR, higher concentrations also lead to neurotransmitter site binding and desensitization. Our results suggest that LAs stabilize multiple conformations of the nAChR by binding to at least two conformationally sensitive LA-binding sites. The spectra also reveal subtle differences in the strengths of the physical interactions that occur between LAs and binding-site residues. These differences correlate with LA potency at the nAChR.

Secondary structure of the exchange-resistant core from the nicotinic acetylcholine receptor probed directly by infrared spectroscopy and hydrogen/deuterium exchange.

The spectral changes that occur in infrared spectra recorded as a function of time after exposure of the nicotinic acetylcholine receptor (nAChR) to 2H2O buffer were examined in order to investigate the secondary structure of the transmembrane domain. The resolution-enhanced amide I band in spectra recorded during the first 12 h after exposure to 2H2O exhibits subtle downshifts in frequency of alpha-helical and beta-sheet vibrations. A strong intensity of the unexchanged alpha-helical vibration near 1655 cm-1 after 3 days exposure to 2H2O suggests that a large proportion of the remaining 25% of unexchanged peptide hydrogens adopts an alpha-helical conformation. Further exposure of the nAChR to 2H2O under conditions of both increasing pH and membrane "fluidity" led to additional exchange of peptide hydrogens for deuterium. The greatest degree of peptide 1H/2H exchange (95%) under nondenaturing conditions was found for the nAChR reconstituted into the highly fluid egg phosphatidylcholine membranes lacking cholesterol and anionic lipids at pH 9.0. This enhanced exchange was accompanied by a decrease in intensity near 1655 cm-1 due to the downshift in frequency of peptides in the alpha-helical conformation, whereas no clear evidence was found for the further exchange of beta-sheet. Some unexchanged alpha-helical peptide hydrogens were still observed. As the exchange-resistant peptides likely include those found within the hydrophobic environment of the lipid bilayer, these data strongly support an alpha-helical secondary structure of the transmembrane domain.

Development of the Logical Observation Identifier Names and Codes (LOINC) vocabulary.

The LOINC (Logical Observation Identifier Names and Codes) vocabulary is a set of more than 10,000 names and codes developed for use as observation identifiers in standardized messages exchanged between clinical computer systems. The goal of the study was to create universal names and codes for clinical observations that could be used by all clinical information systems. The LOINC names are structured to facilitate rapid matching, either automated or manual, between local vocabularies and the universal LOINC codes. If LOINC codes are used in clinical messages, each system participating in data exchange needs to match its local vocabulary to the standard vocabulary only once. This will reduce both the time and cost of implementing standardized interfaces. The history of the development of the LOINC vocabulary and the methodology used in its creation are described.

A cysteine-rich domain of the "mannose" receptor mediates GalNAc-4-SO4 binding.

A critical element of lutropin bioactivity in vivo is its rapid removal from the blood by a receptor, located in hepatic endothelial cells, that recognizes the terminal sulfated carbohydrate structure SO4-4-GalNAcbeta1,4GlcNAcbeta1,2Manalpha (S4GGnM). We have previously shown that the macrophage mannose (Man)-receptor cDNA directs the synthesis of a protein that binds oligosaccharides with either terminal S4GGnM or terminal Man, at independent sites. We now show that the cysteine-rich (Cys-Rich) domain at the N terminus of the Man/S4GGnM receptor accounts for binding of oligosaccharides with terminal GalNAc-4-SO4, whereas calcium-dependent carbohydrate recognition domains (CRDs) account for binding of ligands containing terminal Man. The Cys-Rich domain is thus a previously unrecognized carbohydrate binding motif. Cys-Rich domains have been described on the three other members of the endocytic C-type lectin family of receptors. The structural relationship of these receptors to the Man/S4GGnM receptor raises the possibility that their Cys-Rich domains also bind carbohydrate moieties and contribute to their function.

Secondary structure analysis of individual transmembrane segments of the nicotinic acetylcholine receptor by circular dichroism and Fourier transform infrared spectroscopy.

Circular dichroism (CD) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy are used to establish the secondary structure of peptides containing one or more transmembrane segments (M1-M4) of the Torpedo californica nicotinic acetylcholine receptor (AChR). Peptides containing the M2-M3 and M1-M2-M3 transmembrane segments of the AChR beta-subunit and the M4 segment of the alpha- and gamma-subunits were isolated from proteolytic digests of receptor subunits, purified, and reconstituted into lipid vesicles. For each peptide, an amide I vibrational frequency centered between 1650 and 1656 cm-1 and negative CD absorption bands at 208 and 222 nm indicate that the peptide is largely alpha-helical. In addition, the CD spectrum of a tryptic peptide of the alpha-subunit containing the M1 segment is also consistent with a largely alpha-helical structure. However, secondary structure analysis of the alpha-M1 CD spectrum indicates the presence of other structures, suggesting that the M1 segment may represent either a distorted alpha-helix, likely the consequence of several proline residues, or may not be entirely alpha-helical. Overall, these findings are consistent with studies that indicate that the transmembrane region of the AChR comprises predominantly, if not exclusively, membrane-spanning alpha-helices.