Severe endothelial dysfunction in the aorta of a mouse model of Fabry disease; partial prevention by N-butyldeoxynojirimycin treatment.

OBJECTIVE: Fabry disease results from alpha-gala-ctosidase A deficiency and is characterized by the lysosomal accumulation of globotriaosylceramide. Globotriaosylceramide storage predominantly affects endothelial cells, altering vascular wall morphology and vasomotor function. Our objective was to investigate aortic globotriaosylceramide levels, morphology and function in a mouse model of Fabry disease, and the effect of substrate reduction therapy, using the glycosphingolipid biosynthesis inhibitor N-butyldeoxynojirimycin. METHODS AND RESULTS: Mice used were C57BL/6J and alpha-galactosidase A knockout (Fabry). We show progressive accumulation of aortic globotriaosylceramide throughout the lifespan of untreated Fabry mice (55-fold elevation at 2 months increasing to 187-fold by 19 months), localized to endothelial and vascular smooth-muscle cells; there was no effect on vascular wall morphology in young Fabry mice. In old mice, storage resulted in intimal thickening. Endothelial function declined with age in Fabry mouse aorta. Aortae from N-butyldeoxynojirimycin-treated Fabry mice at 19 months of age had reduced endothelial globotriaosylceramide storage, fewer morphological abnormalities and less severe vasomotor dysfunction compared with untreated littermates. CONCLUSION: We provide evidence of a novel vascular phenotype in the Fabry mouse that has relevance to vascular disease in Fabry patients. N-Butyldeoxynojirimycin treatment partially prevented the phenotype in the Fabry mouse by reducing endothelial globotriaosylceramide storage.

Local and global cerebral blood flow and glucose utilization in the alpha-galactosidase A knockout mouse model of Fabry disease.

Fabry disease is an X-linked lysosomal disorder characterized by deficient alpha-galactosidase A activity and intracellular accumulations of glycosphingolipids, mainly globotriaosylceramide (Gb3). Clinically, patients occasionally present CNS dysfunction. To examine the pathophysiology underlying brain dysfunction, we examined glucose utilization (CMR(glc)) and cerebral blood flow (CBF) globally and locally in 18 brain structures in the alpha-galactosidase A gene knockout mouse. Global CMR(glc) was statistically significantly reduced by 22% in Fabry mice (p < 0.01). All 18 structures showed decreases in local CMR(glc) ranging from 14% to 33%. The decreases in all structures of the diencephalon, caudate-putamen, brain stem, and cerebellar cortex were statistically significant (p < 0.05). Global cerebral blood flow (CBF) and local CBF measured in the same 18 structures were lower in Fabry mice than in control mice, but none statistically significantly. Histological examination of brain revealed no cerebral infarcts but abundant Gb3 deposits in the walls of the cerebral vessels with neuronal deposits localized to the medulla oblongata. These results indicate an impairment in cerebral energy metabolism in the Fabry mice, but one not necessarily due to circulatory insufficiency.

alpha-Lactalbumin (LA) stimulates milk beta-1,4-galactosyltransferase I (beta 4Gal-T1) to transfer glucose from UDP-glucose to N-acetylglucosamine. Crystal structure of beta 4Gal-T1 x LA complex with UDP-Glc.

beta-1,4-Galactosyltransferase 1 (Gal-T1) transfers galactose (Gal) from UDP-Gal to N-acetylglucosamine (GlcNAc), which constitutes its normal galactosyltransferase (Gal-T) activity. In the presence of alpha-lactalbumin (LA), it transfers Gal to Glc, which is its lactose synthase (LS) activity. It also transfers glucose (Glc) from UDP-Glc to GlcNAc, constituting the glucosyltransferase (Glc-T) activity, albeit at an efficiency of only 0.3-0.4% of Gal-T activity. In the present study, we show that LA increases this activity almost 30-fold. It also enhances the Glc-T activity toward various N-acyl substituted glucosamine acceptors. Steady state kinetic studies of Glc-T reaction show that the K(m) for the donor and acceptor substrates are high in the absence of LA. In the presence of LA, the K(m) for the acceptor substrate is reduced 30-fold, whereas for UDP-Glc it is reduced only 5-fold. In order to understand this property, we have determined the crystal structures of the Gal-T1.LA complex with UDP-Glc x Mn(2+) and with N-butanoyl-glucosamine (N-butanoyl-GlcN), a preferred sugar acceptor in the Glc-T activity. The crystal structures reveal that although the binding of UDP-Glc is quite similar to UDP-Gal, there are few significant differences observed in the hydrogen bonding interactions between UDP-Glc and Gal-T1. Based on the present kinetic and crystal structural studies, a possible explanation for the role of LA in the Glc-T activity has been proposed.

Crystal structure of lactose synthase reveals a large conformational change in its catalytic component, the beta1,4-galactosyltransferase-I.

The lactose synthase (LS) enzyme is a 1:1 complex of a catalytic component, beta1,4-galactosyltransferse (beta4Gal-T1) and a regulatory component, alpha-lactalbumin (LA), a mammary gland-specific protein. LA promotes the binding of glucose (Glc) to beta4Gal-T1, thereby altering its sugar acceptor specificity from N-acetylglucosamine (GlcNAc) to glucose, which enables LS to synthesize lactose, the major carbohydrate component of milk. The crystal structures of LS bound with various substrates were solved at 2 A resolution. These structures reveal that upon substrate binding to beta4Gal-T1, a large conformational change occurs in the region comprising residues 345 to 365. This repositions His347 in such a way that it can participate in the coordination of a metal ion, and creates a sugar and LA-binding site. At the sugar-acceptor binding site, a hydrophobic N-acetyl group-binding pocket is found, formed by residues Arg359, Phe360 and Ile363. In the Glc-bound structure, this hydrophobic pocket is absent. For the binding of Glc to LS, a reorientation of the Arg359 side-chain occurs, which blocks the hydrophobic pocket and maximizes the interactions with the Glc molecule. Thus, the role of LA is to hold Glc by hydrogen bonding with the O-1 hydroxyl group in the acceptor-binding site on beta4Gal-T1, while the N-acetyl group-binding pocket in beta4Gal-T1 adjusts to maximize the interactions with the Glc molecule. This study provides details of a structural basis for the partially ordered kinetic mechanism proposed for lactose synthase.

Glycolipid antigen processing for presentation by CD1d molecules.

The requirement for processing glycolipid antigens in T cell recognition was examined with mouse CD1d-mediated responses to glycosphingolipids (GSLs). Although some disaccharide GSL antigens can be recognized without processing, the responses to three other antigens, including the disaccharide GSL Gal(alpha1-->2)GalCer (Gal, galactose; GalCer, galactosylceramide), required removal of the terminal sugars to permit interaction with the T cell receptor. A lysosomal enzyme, alpha-galactosidase A, was responsible for the processing of Gal(alpha1-->2)GalCer to generate the antigenic monosaccharide epitope. These data demonstrate a carbohydrate antigen processing system analogous to that used for peptides and an ability of T cells to recognize processed fragments of complex glycolipids.

Adeno-associated viral vector-mediated gene transfer results in long-term enzymatic and functional correction in multiple organs of Fabry mice.

Fabry disease is a lysosomal storage disorder caused by a deficiency of the lysosomal enzyme alpha-galactosidase A (alpha-gal A). This enzyme deficiency leads to impaired catabolism of alpha-galactosyl-terminal lipids such as globotriaosylceramide (Gb3). Patients develop painful neuropathy and vascular occlusions that progressively lead to cardiovascular, cerebrovascular, and renal dysfunction and early death. Although enzyme replacement therapy and bone marrow transplantation have shown promise in the murine analog of Fabry disease, gene therapy holds a strong potential for treating this disease in humans. Delivery of the normal alpha-gal A gene (cDNA) into a depot organ such as liver may be sufficient to elicit corrective circulating levels of the deficient enzyme. To investigate this possibility, a recombinant adeno-associated viral vector encoding human alpha-gal A (rAAV-AGA) was constructed and injected into the hepatic portal vein of Fabry mice. Two weeks postinjection, alpha-gal A activity in the livers of rAAV-AGA-injected Fabry mice was 20-35% of that of the normal mice. The transduced animals continued to show higher alpha-gal A levels in liver and other tissues compared with the untouched Fabry controls as long as 6 months after treatment. In parallel to the elevated enzyme levels, we see significant reductions in Gb3 levels to near normal at 2 and 5 weeks posttreatment. The lower Gb3 levels continued in liver, spleen, and heart, up to 25 weeks with no significant immune response to the virus or alpha-gal A. Also, no signs of liver toxicity occurred after the rAAV-AGA administration. These findings suggest that an AAV-mediated gene transfer may be useful for the treatment of Fabry disease and possibly other metabolic disorders.

Active site studies of bovine alpha1-->3-galactosyltransferase and its secondary structure prediction.

The catalytic domain of bovine alpha1-->3-galactosyltransferase (alpha3GalT), residues 80-368, have been cloned and expressed, in Escherichia coli. Using a sequential purification protocol involving a Ni(2+) affinity column followed by a UDP-hexanolamine affinity column, we have obtained a pure and active protein from the soluble fraction which catalyzes the transfer of galactose (Gal) from UDP-Gal to N-acetyllactosamine (LacNAc) with a specific activity of 0.69 pmol/min/ng. The secondary structural content of alpha3GalT protein was analyzed by Fourier transform infrared (FTIR) spectroscopy, which shows that the enzyme has about 35% beta-sheet and 22% alpha-helix. This predicted secondary structure content by FTIR spectroscopy was used in the protein sequence analysis algorithm, developed by the Biomolecular Engineering Research Center at Boston University and Tasc Inc., for the assignment of secondary structural elements to the amino acid sequence of alpha3GalT. The enzyme appears to have three major and three minor helices and five sheet-like structures. The studies on the acceptor substrate specificity of the enzyme, alpha3GalT, show that in addition to LacNAc, which is the natural substrate, the enzyme accepts various other disaccharides as substrates such as lactose and Gal derivatives, beta-O-methylgalactose and beta-D-thiogalactopyranoside, albeit with lower specific activities. There is an absolute requirement for Gal to be at the non-reducing end of the acceptor molecule which has to be beta1-->4-linked to a second residue that can be more diverse in structure. The kinetic parameters for four acceptor molecules were determined. Lactose binds and functions in a similar way as LacNAc. However, beta-O-methylgalactose and Gal do not bind as tightly as LacNAc or lactose, as their K(ia) and K(A) values indicate, suggesting that the second monosaccharide is critical for holding the acceptor molecule in place. The 2' and 4' hydroxyl groups of the receiving Gal moiety are important in binding. Even though there is large structural variability associated with the second residue of the acceptor molecule, there are constraints which do not allow certain Gal-R sugars to be good acceptors for the enzyme. The beta1-->4-linked residue at the second position of the acceptor molecule is preferred, but the interactions between the enzyme and the second residue are likely to be non-specific.

Long-term enzyme correction and lipid reduction in multiple organs of primary and secondary transplanted Fabry mice receiving transduced bone marrow cells.

Fabry disease is a compelling target for gene therapy as a treatment strategy. A deficiency in the lysosomal hydrolase alpha-galactosidase A (alpha-gal A; EC ) leads to impaired catabolism of alpha-galactosyl-terminal lipids such as globotriaosylceramide (Gb3). Patients develop vascular occlusions that cause cardiovascular, cerebrovascular, and renal disease. Unlike for some lysosomal storage disorders, there is limited primary nervous system involvement in Fabry disease. The enzyme defect can be corrected by gene transfer. Overexpression of alpha-gal A by transduced cells results in secretion of this enzyme. Secreted enzyme is available for uptake by nontransduced cells presumably by receptor-mediated endocytosis. Correction of bystander cells may occur locally or systemically after circulation of the enzyme in the blood. In this paper we report studies on long-term genetic correction in an alpha-gal A-deficient mouse model of Fabry disease. alpha-gal A-deficient bone marrow mononuclear cells (BMMCs) were transduced with a retrovirus encoding alpha-gal A and transplanted into sublethally and lethally irradiated alpha-gal A-deficient mice. alpha-gal A activity and Gb3 levels were analyzed in plasma, peripheral blood mononuclear cells, BMMCs, liver, spleen, heart, lung, kidney, and brain. Primary recipient animals were followed for up to 26 weeks. BMMCs were then transplanted into secondary recipients. Increased alpha-gal A activity and decreased Gb3 storage were observed in all recipient groups in all organs and tissues except the brain. These effects occurred even with a low percentage of transduced cells. The findings indicate that genetic correction of bone marrow cells derived from patients with Fabry disease may have utility for phenotypic correction of patients with this disorder.

Human mesenchymal stem cells support megakaryocyte and pro-platelet formation from CD34(+) hematopoietic progenitor cells.

Megakaryocytopoiesis and thrombocytopoiesis result from the interactions between hematopoietic progenitor cells, humoral factors, and marrow stromal cells derived from mesenchymal stem cells (MSCs) or MSCs directly. MSCs are self-renewing marrow cells that provide progenitors for osteoblasts, adipocytes, chondrocytes, myocytes, and marrow stromal cells. MSCs are isolated from bone marrow aspirates and are expanded in adherent cell culture using an optimized media preparation. Culture-expanded human MSCs (hMSCs) express a variety of hematopoietic cytokines and growth factors and maintain long-term culture-initiating cells in long-term marrow culture with CD34(+) hematopoietic progenitor cells. Two lines of evidence suggest that hMSCs function in megakaryocyte development. First, hMSCs express messenger RNA for thrombopoietin, a primary regulator for megakaryocytopoiesis and thrombocytopoiesis. Second, adherent hMSC colonies in primary culture are often associated with hematopoietic cell clusters containing CD41(+) megakaryocytes. The physical association between hMSCs and megakaryocytes in marrow was confirmed by experiments in which hMSCs were copurified by immunoselection using an anti-CD41 antibody. To determine whether hMSCs can support megakaryocyte and platelet formation in vitro, we established a coculture system of hMSCs and CD34(+) cells in serum-free media without exogenous cytokines. These cocultures produced clusters of hematopoietic cells atop adherent MSCs. After 7 days, CD41(+) megakaryocyte clusters and pro-platelet networks were observed with pro-platelets increasing in the next 2 weeks. CD41(+) platelets were found in culture medium and expressed CD62P after thrombin treatment. These results suggest that MSCs residing within the megakaryocytic microenvironment in bone marrow provide key signals to stimulate megakaryocyte and platelet production from CD34(+) hematopoietic cells.

Molecular dynamics simulation of alpha-lactalbumin and calcium binding c-type lysozyme.

Alpha-lactalbumins (LAs) and c-type lysozymes (LYZs) are two classes of proteins which have a 35-40% sequence homology and share a common three dimensional fold but perform different functions. Lysozymes bind and cleave the glycosidic bond linkage in sugars, where as, alpha-lactalbumin does not bind sugar but participates in the synthesis of lactose. Alpha-lactalbumin is a metallo-protein and binds calcium, where as, only a few of the LYZs bind calcium. These proteins consist of two domains, an alpha-helical and a beta-strand domain, separated by a cleft. Calcium is bound at a loop situated at the bottom of the cleft and is important for the structural integrity of the protein. Calcium is an ubiquitous intracellular signal in higher eukaryotes and structural changes induced on calcium binding have been observed in a number of proteins. In the present study, molecular dynamics simulations of equine LYZ and human LA, with and without calcium, were carried out. We detail the differences in the dynamics of equine LYZ and human LA, and discuss it in the light of experimental data already available and relate it to the behavior of the functionally important regions of both the proteins. These simulations bring out the role of calcium in the conformation and dynamics of these metallo-proteins. In the calcium bound LA, the region of the protein around the calcium binding site is not only frozen but the atomic fluctuations are found to increase away from the binding site and peak at the exposed sites of the protein. This channeling of fluctuations away from the metal binding site could serve as a general mechanism by which the effect of metal binding at a site is transduced to other parts of the protein and could play a key role in protein-ligand and/or protein-protein interaction.

Architecture of the sugar binding sites in carbohydrate binding proteins--a computer modeling study.

Different sugars, Gal, GalNAc and Man were docked at the monosaccharide binding sites of Erythrina corallodenron (EcorL), peanut lectin (PNA), Lathyrus ochrus (LOLI), and pea lectin (PSL). To study the lectin-carbohydrate interactions, in the complexes, the hydroxymethyl group in Man and Gal favors, gg and gt conformations respectively, and is the dominant recognition determination. The monosaccharide binding site in lectins that are specific to Gal/GalNAc is wider due to the additional amino acid residues in loop D as compared to that in lectins specific to Man/Glc, and affects the hydrogen bonds of the sugar involving residues from loop D, but not its orientation in the binding site. The invariant amino acid residues Asp from loop A, and Asn and an aromatic residue (Phe or Tyr) in loop C provides the basic architecture to recognize the common features in C4 epimers. The invariant Gly in loop B together with one or two residues in the variable region of loop D/A holds the sugar tightly at both ends. Loss of any one of these hydrogen bonds leads to weak interaction. While the subtle variations in the sequence and conformation of peptide fragment that resulted due to the size and location of gaps present in amino acid sequence in the neighborhood of the sugar binding site of loop D/A seems to discriminate the binding of sugars which differ at C4 atom (galacto and gluco configurations). The variations at loop B are important in discriminating Gal and GalNAc binding. The present study thus provides a structural basis for the observed specificities of legume lectins which uses the same four invariant residues for binding. These studies also bring out the information that is important for the design/engineering of proteins with the desired carbohydrate specificity.

Molecular modeling studies on binding of bFGF to heparin and its receptor FGFR1.

Sugar induced protein-protein interactions play an important role in several biological processes. The carbohydrate moieties of proteoglycans, the glycosaminoglycans, bind to growth factors with a high degree of specificity and induce interactions with growth factor receptors, thereby regulate the growth factor activity. We have used molecular modeling method to study the modes of binding of heparin or heparan sulfate proteoglycans (HSPGs) to bFGF that leads to the dimerization of FGF receptor 1 (FGFR1) and activation of receptor tyrosine kinase. Homology model of FGFR1 Ig D(II)-D(III) domains was built to investigate the interactions between heparin, bFGF and FGFR1. The structural requirements to bridge the two monomeric bFGF molecules by heparin or HSPGs and to simulate the dimerization and activation of FGFR1 have been examined. A structural model of the biologically functional dimeric bFGF-heparin complex is proposed based on: (a) the stability of dimeric complex, (b) the favorable binding energies between heparin and bFGF molecules, and (c) its accessibility to FGFR1. The modeled complex between heparin, bFGF and FGFR1 has a stoichiometry of 1 heparin: 2 bFGF: 2 FGFR1. The structural properties of the proposed model of bFGF/heparin/FGFR1 complex are consistent with the binding mechanism of FGF to its receptor, the receptor dimerization, and the reported site-specific mutagenesis and biochemical cross-linking data. In the proposed model heparin bridges the two bFGF monomers in a specific orientation and the resulting complex induces FGF receptor dimerization, suggesting that in the oligosaccharide induced recognition process sugars orient the molecules in a way that brings about specific protein-protein or protein-carbohydrate interactions.

Three dimensional structure of the soybean agglutinin Gal/GalNAc complexes by homology modeling.

Complexes of soybean agglutinin (SBA) with galactose (Gal) and N-acetyl galactosamine (GalNAc) have been modeled based on its homology to erythrina corallodendron (EcorL) lectin. The three dimensional structure of SBA-Gal modeled with homology techniques agrees well with SBA-(beta-LacNAc)2Gal-R complex determined by X-ray crystallographic techniques at the beta-sheet regions and the regions where Ca2+ and Mn2+ ions bind. However, significant deviations have been observed between the modeled and the X-ray structures, particularly at the loop regions where the polypeptide chain could not be unequivocally traced in the X-ray structure. The hydrogen bonding scheme, predicted from the homology model, shows that the invariant residues i.e. Asp, Gly, Asn, and aromatic residues (Phe) found in all other legume lectins, bind Gal, slightly in a different way than reported in X-ray structure of SBA-pentasaccharide complex. The higher binding affinity of GalNAc over Gal to SBA is due to additional hydrophobic interactions with Tyr107 rather than a hydrogen bond between N-acetamide group of the sugar and the side chain of Asp88 as suggested from X-ray crystal structure studies. Our modeling also suggest that the variation in the length of the loop D observed among galactose binding legume lectins may not have any effect on the binding of sugar at the monosaccharide specific site of the lectins. Soybean agglutinin (SBA) is a member of the leguminous family of lectins. They generally possess a single carbohydrate binding site, besides the tightly bound Ca2+ and Mn2+ ions which are required for their carbohydrate binding activity. They possess a high degree of sequence homology and about 50% of the amino acid residues are invariant. Some of these invariant amino acid residues are involved in the binding of sugar moieties and in metal ion coordination. X-ray crystallographic studies showed that their three-dimensional structures are very similar, though they differ in their carbohydrate binding specificity (1-6). Three of the invariant residues Asp, Gly, and Asn, besides an aromatic residue (Phe or Tyr), are involved in carbohydrate binding. Independent of their sugar specificity, these four residues in legume lectins provide the basic frame for the sugar to bind.

Tissue and zonal-specific expression of an olfactory receptor transgene.

Discrimination of odorants is thought to arise from the selective expression of one of a small number of individual receptors in any single olfactory neuron. Receptor genes are expressed in a small subset of neurons throughout a zonally restricted region of the sensory epithelium. We demonstrate that a 6.7 kb region upstream of the M4 olfactory receptor coding region was sufficient to direct expression in olfactory epithelium. Moreover, reporter expression recapitulated the zonal restriction and distributed neuronal expression observed for endogenous olfactory receptors. Transgenic lines were obtained that directed expression in two different receptor zones, one of which was identical to the endogenous M4 receptor. When the reporter was expressed in the same zone as the endogenous M4 receptor, the two expression patterns were, in large part, nonoverlapping. These results suggest a model in which important regulatory elements are located in close proximity to transcription initiation sites of the olfactory receptor genes and receive information defining zonal patterning via long-range processes.

Putative odour receptors localize in cilia of olfactory receptor cells in rat and mouse: a freeze-substitution ultrastructural study.

Two different polyclonal antibodies were raised to synthetic peptides corresponding to distinct putative odour receptors of rat and mouse. Both antibodies selectively labelled olfactory cilia as seen with cryofixation and immunogold ultrastructural procedures. Regions of the olfactory organ where label was detected were consistent with those found at LM levels. Immunopositive cells were rare; only up to about 0.4% of these receptor cells were labelled. Despite chemical, species, and topographic differences both antibodies behaved identically in their ultrastructural labelling patterns. For both antibodies, labelling was very specific for olfactory cilia; both bound amply to the thick proximal and the thinner and long distal parts of the cilia. Dendritic knobs showed little labelling if any. Dendritic receptor cell structures below the knobs, supporting cell structures, and respiratory cilia did not immunolabel. There were no obvious differences in morphology between labelled and unlabelled receptor cells and their cilia. Labelling could be followed up to a distance of about 15 microns from the knobs along the distal parts of the cilia. When labelled cells were observed, this signal was detectable in two, sometimes three, sections taken through these cells while being consistently absent in neighbouring cells. This pattern argues strongly for the specificity of the labelling. In conclusion, very few receptor cells labelled with the antibodies to putative odour receptors. Additionally the olfactory cilia, the cellular regions that first encounter odour molecules and that are thought to transduce the odorous signal, displayed the most intense labelling with both antibodies. Consequently, the results showed these cilia as having many copies of the putative receptors. Finally, similar patterns of subcellular labelling were displayed in two different species, despite the use of different antibodies. Thus, this study provides compelling evidence that the heptahelical putative odour receptors localize in the olfactory cilia.

Putative odour receptors localize in cilia of olfactory receptor cells in rat and mouse: a freeze-substitution ultrastructural study.

Two different polyclonal antibodies were raised to synthetic peptides corresponding to distinct putative odour receptors of rat and mouse. Both antibodies selectively labelled olfactory cilia as seen with cryofixation and immunogold ultrastructural procedures. Regions of the olfactory organ where label was detected were consistent with those found at LM levels. Immunopositive cells were rare; only up to about 0.4% of these receptor cells were labelled. Despite chemical, species, and topographic differences both antibodies behaved identically in their ultrastructural labelling patterns. For both antibodies, labelling was very specific for olfactory cilia; both bound amply to the thick proximal and the thinner and long distal parts of the cilia. Dendritic knobs showed little labelling if any. Dendritic receptor cell structures below the knobs, supporting cell structures, and respiratory cilia did not immunolabel. There were no obvious differences in morphology between labelled and unlabelled receptor cells and their cilia. Labelling could be followed up to a distance of about 15 microns from the knobs along the distal parts of the cilia. When labelled cells were observed, this signal was detectable in two, sometimes three, sections taken through these cells while being consistently absent in neighbouring cells. This pattern argues strongly for the specificity of the labelling. In conclusion, very few receptor cells labelled with the antibodies to putative odour receptors. Additionally the olfactory cilia, the cellular regions that first encounter odour molecules and that are thought to transduce the odorous signal, displayed the most intense labelling with both antibodies. Consequently, the results showed these cilia as having many copies of the putative receptors. Finally, similar patterns of subcellular labelling were displayed in two different species, despite the use of different antibodies. Thus, this study provides compelling evidence that the heptahelical putative odour receptors localize in the olfactory cilia.

Molecular divergence of lysozymes and alpha-lactalbumin.

The vast number of proteins that sustain the currently living organisms have been generated from a relatively small number of ancestral genes that has involved a variety of processes. Lysozyme is an ancient protein whose origin goes back an estimated 400 to 600 million years. This protein was originally a bacteriolytic defensive agent and has been adapted to serve a digestive function on at least two occasions, separated by nearly 40 million years. The origins of the related goose type and T4 phage lysozyme that are distinct from the more common C type are obscure. They share no discernable amino acid sequence identity and yet they possess common secondary and tertiary structures. Lysozyme C gene also gave rise, after gene duplication 300 to 400 million years ago, to a gene that currently codes for alpha-lactalbumin, a protein expressed only in the lactating mammary gland of all but a few species of mammals. It is required for the synthesis of lactose, the sugar secreted in milk. alpha-Lactalbumin shares only 40% identity in amino acid sequence with lysozyme C, but it has a closer spatial structure and gene organization. Although structurally similar, functionally they are quite distinct. Specific amino acid substitutions in alpha-lactalbumin account for the loss of the enzyme activity of lysozyme and the acquisition of the features necessary for its role in lactose synthesis. Evolutionary implications are as yet unclear but are being unraveled in many laboratories.

Molecular dynamics simulations of hybrid and complex type oligosaccharides.

Conformational preferences of hybrid (GlcNAc1Man5GlcNAc2) and complex (GlcNAc1Man3GlcNAc2; GlcNAc2Man3GlcNAc2) type asparagine-linked oligosaccharides and the corresponding bisected oligosaccharides have been studied by molecular dynamics simulations for 2.5 ns. The fluctuations of the core Man-alpha 1,3-Man fragment are restricted to a region around (-30 degrees, -30 degrees) due to a 'face-to-face' arrangement of bisecting GlcNAc and the beta 1,2-GlcNAc on the 1,3-arm. However, conformations where such a 'face-to-face' arrangement is disrupted are also accessed occasionally. The orientation of the 1,6-arm is affected not only by changes in chi, but also by changes in phi and psi around the core Man-alpha 1,6-Man linkage. The conformation around the core Man-alpha 1,6-Man linkage is different in the hybrid and the two complex types suggesting that the preferred values of phi, psi, and chi are affected by the addition or deletion of saccharides to the alpha 1,6-linked mannose. The conformational data are in agreement with the available experimental studies and also explain the branch specificity of galactosyltransferases.

Functional domains of bovine beta-1,4 galactosyltransferase.

A number of N- and C-terminal deletion and point mutants of bovine beta-1,4 galactosyltransferase (beta-1,4GT) were expressed in E. coli to determine the binding regions of the enzyme that interact with N-acetylglucosamine (NAG) and UDP-galactose. The N-terminal truncated forms of the enzyme between residues 1-129, do not show any significant difference in the apparent Kms towards NAG or linear oligosaccharide acceptors e.g. for chitobiose and chitotriose, or for the nucleotide donor UDP-galactose. Deletion or mutation of Cys 134 results in the loss of enzymatic activity, but does not affect the binding properties of the protein either to NAG- or UDP-agarose. From these columns the protein can be eluted with 15 mM NAG and 50 mM EDTA, like the enzymatically active protein, TL-GT129, that contains residues 130-402 of bovine beta-1,4GT. Also the N-terminus fragment, TL-GT129NAG, that contains residues 130-257 of the beta-1,4GT, binds to, and elutes with 15 mM NAG and 50 mM EDTA from the NAG-agarose column as efficiently as the enzymatically active TL-GT129. Unlike TL-GT129, the TL-GT129NAG binds to UDP-columns less efficiently and can be eluted from the column with only 15 mM NAG. The C-terminus fragment GT-257UDP, containing residues 258-402 of beta-1,4GT, binds tightly to both NAG- and UDP-agarose columns. A small fraction, 5-10% of the bound protein, can be eluted from the UDP-agarose column with 50 mM EDTA alone. The results show that the binding behaviour of N- and C-terminal fragments of beta-1,4GT towards the NAG- and UDP-agarose columns differ, the former binds preferentially to NAG-columns, while the latter binds to UDP-agarose columns via Mn2+.