Oligosaccharide analysis and molecular modeling of soluble forms of glycoproteins belonging to the Ly-6, scavenger receptor, and immunoglobulin superfamilies expressed in Chinese hamster ovary cells

Most cell surface molecules are glycoproteins consisting of linear arrays of globular domains containing stretches of amino acid sequence with similarities to regions in other proteins. These conserved regions form the basis for the classification of proteins into superfamilies. Recombinant soluble forms of six leukocyte antigens belonging to the Ly-6 (CD59), scavenger receptor (CD5), and immunoglobulin (CD2, CD48, CD4, and Thy-1) superfamilies were expressed in the same Chinese hamster ovary cell line, thus providing an opportunity to examine the extent to which N-linked oligosaccharide processing might vary in a superfamily-, domain-, or protein-dependent manner in a given cell. While we found no evidence for superfamily-specific modifications of the glycans, marked differences were seen in the types of oligosaccharides attached to individual proteins within a given superfamily. The relative importance of local protein surface properties versus the overall tertiary structure of the molecules in directing this protein-specific variation was examined in the context of molecular models. These were constructed using the 3D structures of the proteins, glycan data from this study, and an oligosaccharide structural database. The results indicated that both the overall organization of the domains and the local protein structure can have a large bearing on site-specific glycan modification of cells in stasis. This level of control ensures that the surface of a single cell will display a diverse repertoire of glycans and precludes the presentation of multiple copies of a single oligosaccharide on the cell surface. The glycans invariably shield large regions of the protein surfaces although, for the glycoproteins examined here, these did not hinder the known active sites of the molecules. The models also indicated that sugars are likely to play a role in the packing of the native cell surface glycoproteins and to limit nonspecific protein-protein interactions. In addition, glycans located close to the cell membrane are likely to affect crucially the orientation of the glycoproteins to which they are attached.     

Synthesis of fluorescence-labelled disaccharide substrates of glucosidase II

The fluorescence-labelled disaccharides Glcalpha(1-->3)GlcalphaOR and Glcalpha(1-->3)ManalphaOR, both substrates for the glycoprotein-processing enzyme glucosidase II, were synthesised via the use of a n-pentenyl-derived linker at the anomeric position. This allowed incorporation of a pyrenebutyric acid label, via a sequence of oxidative hydroboration, mesylation, azide displacement, reduction with concomitant global deprotection, and peptide coupling. Selective activation of a fully armed thioglycoside in the presence of n-pentenyl glycosides was readily achieved by the use of methyl triflate as promoter.     

Metabolomics as a diagnostic tool for hepatology: validation in a naturally occurring canine model

Human hepatopathies are a diagnostic challenge, with many distinct diseases having similar clinical signs and laboratory findings. Naturally occurring canine hepatic disease provides an excellent model for human diseases and similar diagnostic dilemmas exist; differentiating canine congenital portosystemic vascular anomalies (PVA) from acquired hepatopathies is difficult and traditionally requires invasive diagnostic procedures. The emerging post-genomic science of metabolomics is concerned with detecting global changes of populations of endogenous low molecular weight metabolites in biological samples and offers the possibility of identifying surrogate profiles of disease. Metabolomics couples sensitive metabolite analysis with sophisticated pattern recognition techniques. In this study, a metabolomic strategy has been employed to assess metabolite changes in the plasma of dogs with congenital PVA and acquired hepatic disease. Plasma samples were collected from 25 dogs, comprising 9 dogs with congenital PVA, 6 with acquired hepatopathy and 10 with non-hepatic disorders. Low molecular weight metabolites were analyzed by liquid chromatography-mass spectrometry (LC-MS). Following identification of metabolites, multivariate data analysis was used to compare profiles amongst groups. The analysis demonstrated significant disturbances in the plasma bile acid and phospholipid profiles of dogs with portovascular anomalies. In contrast to traditional laboratory parameters, the metabolomic strategy was able to produce a clear segregation between all three study groups. In conclusion, this study demonstrates the potential of metabolomics as a diagnostic tool for naturally occurring hepatic disease. With further validation, this approach will improve diagnostic capabilities, provide an insight into pathogenetic mechanisms, and ultimately inform therapeutic decision making in clinical hepatology. This work was supported in part by grants from The Royal Society, Petplan Charitable Trust and The Waltham Foundation.     

maternally expressed gene1 Is a novel maize endosperm transfer cell-specific gene with a maternal parent-of-origin pattern of expression

Growth of the maize (Zea mays) endosperm is tightly regulated by maternal zygotic and sporophytic genes, some of which are subject to a parent-of-origin effect. We report here a novel gene, maternally expressed gene1 (meg1), which shows a maternal parent-of-origin expression pattern during early stages of endosperm development but biallelic expression at later stages. Interestingly, a stable reporter fusion containing the meg1 promoter exhibits a similar pattern of expression. meg1 is exclusively expressed in the basal transfer region of the endosperm. Further, we show that the putatively processed MEG1 protein is glycosylated and subsequently localized to the labyrinthine ingrowths of the transfer cell walls. Hence, the discovery of a parent-of-origin gene expressed solely in the basal transfer region opens the door to epigenetic mechanisms operating in the endosperm to regulate certain aspects of nutrient trafficking from the maternal tissue into the developing seed.     

Endurance training partially reverses dietary-induced leptin resistance in rodent skeletal muscle

Leptin acutely stimulates skeletal muscle fatty acid (FA) metabolism in lean rodents and humans. This stimulatory effect is eliminated following the feeding of high-fat diets in rodents as well as in obese humans. The mechanism(s) responsible for the development of skeletal muscle leptin resistance is unknown; however, a role for increased suppressor of cytokine signaling-3 (SOCS3) inhibition of the leptin receptor has been demonstrated in other rodent tissues. Furthermore, whether exercise intervention is an effective strategy to prevent or attenuate the development of skeletal muscle leptin resistance has not been investigated. Toward this end, 48 Sprague-Dawley rats (175-190 g; approximately 2-3 mo of age) were fed control or high-fat (60% kcal) diets for 4 wk and either remained sedentary or were treadmill trained. In control diet-fed animals that remained sedentary (CS) or were endurance trained (CT), leptin stimulated FA oxidation (CS +32 +/- 15%, CT +30 +/- 17%; P < 0.05), suppressed triacylglycerol (TAG) esterification (CS -17 +/- 7%, CT -24 +/- 8%; P < 0.05), and reduced the esterification-to-oxidation ratio (CS -19 +/- 13%, CT -29 +/- 10%; P < 0.001) in soleus muscle. High-fat feeding induced leptin resistance in the soleus of sedentary rats (FS), whereas endurance exercise training (FT) restored the ability of leptin to suppress TAG esterification (-19 +/- 9%, P = 0.038). Training did not completely restore the ability of leptin to stimulate FA oxidation. High-fat diets stimulated SOCS3 mRNA expression irrespective of training status (FS +451 +/- 120%, P = 0.024; FT +381 +/- 141%, P = 0.023). Thus the development of skeletal muscle leptin resistance appears to involve an increase in SOCS3 mRNA expression. Endurance training was generally effective in preventing the development of leptin resistance, although this did not appear to require a decrease in SOCS3 expression. Future studies should examine changes in the actual protein content of SOCS3 in muscle and establish whether aerobic exercise is also effective in treating leptin resistance in humans.     

Afamin is a novel human vitamin E-binding glycoprotein characterization and in vitro expression

Hydrophobic vitamins are transported in human plasma and extravascular fluids by carrier proteins. No specific protein has been described so far for vitamin E, which plays a crucial role in protecting against oxidative damage and disease. We report here the purification of a 75-kDa glycoprotein with vitamin E-binding properties by stepwise chromatography of lipoprotein-depleted human plasma and monitoring of vitamin E (alpha-tocopherol)-binding activity. Partial sequencing identified this protein as afamin, a previously described member of the albumin gene family with four or five potential N-glycosylation sites. Glycosylation analysis indicated that >90% of the glycans were sialylated biantennary complex structures. The vitamin E-binding properties were confirmed using recombinantly expressed afamin. Qualitative and quantitative analysis of plasma and extravascular fluids revealed an abundant presence of this protein not only in plasma (59.8+/-13.3 microg/mL) but also in extravascular fluids such as follicular (34.4+/-12.7 microg/mL) and cerebrospinal (0.28+/-0.16 microg/mL) fluids, suggesting potential roles for afamin in fertility and neuroprotection. Afamin is partly (13%) bound to plasma lipoproteins. Afamin and vitamin E concentrations significantly correlate in follicular and cerebrospinal fluids but not in plasma. The vitamin E association of afamin in follicular fluid was directly demonstrated by gel filtration chromatography and immunoprecipitation which complements the in vitro findings for purified native and recombinant afamin.     

Hybrid and complex glycans are linked to the conserved N-glycosylation site of the third eight-cysteine domain of LTBP-1 in insect cells

Covalent association of LTBP-1 (latent TGF-beta binding protein-1) to latent TGF-beta is mediated by the third eight-cysteine (also referred to as TB) module of LTBP-1, a domain designated as CR3. Spodoptera frugiperda (Sf9) cells have proved a suitable cell system in which to study this association and to produce recombinant CR3, and we show here that another lepidopteran cell line, Trichoplusia niTN-5B1-4 (High-Five) cells, allows the recovery of large amounts of functional recombinant CR3. CR3 contains an N-glycosylation site, which is conserved in all forms of LTBP known to date. When we examined the status of this N-glycosylation using MALDI-TOF mass spectrometry and enzymatic analysis, we found that CR3 is one of the rare recombinant peptides modified with complex glycans in insect cells. Sf9 cells mainly processed the fucosylated paucomannosidic structure (GlcNAc)(2)(Mannose)(3)Fucose, although hybrid and complex N-glycosylations were also detected. In High-Five cells, the peptide was found to be modified with a wide variety of hybrid and complex sugars in addition to paucomanosidic oligosaccharides. Most glycans had one or two fucose residues bound through alpha1,3 and alpha1,6 linkages to the innermost GlcNAc. On the basis of these results and on the structure of an eight-cysteine domain from fibrillin-1, we present a model of glycosylated CR3 and discuss the role of glycosylation in eight-cysteine domain protein-protein interactions.     

Solution NMR studies of colicin E1 C-terminal thermolytic peptide. Structural comparison with colicin A and the effects of pH changes

The aqueous solution structure of the C-terminal thermolytic peptide of colicin E1 has been investigated using both one- and two-dimensional NMR techniques. The NMR data are consistent with a fold for the peptide very similar to that reported for the colicin A C-terminal peptide in the crystalline state, although some differences have been noted. The one-dimensional NMR spectrum of the peptide has been used to follow changes in both the structure and dynamics of the peptide on changing pH. The in vitro functionally competent form of the peptide (present in solution only below pH 6) does not differ in structure significantly from the higher pH form. However, small local conformational changes are observed together with an increase in mobility in some of the more hydrophilic regions. This suggests that the effect of lower pH is to change the ease with which the major conformational changes during insertion into a membrane can occur.