No abstract

Mass spectrometry (MS) of glycoproteins is an emerging field in proteomics, poised to meet the technical demand for elucidation of the structural complexity and functions of the oligosaccharide components of molecules. Considering the divergence of the mass spectrometric methods employed for oligosaccharide analysis in recent publications, it is necessary to establish technical standards and demonstrate capabilities. In the present study of the Human Proteome Organisation (HUPO) Human Disease Glycomics/Proteome Initiative (HGPI), the same samples of transferrin and immunoglobulin-G were analyzed for N-linked oligosaccharides and their relative abundances in 20 laboratories, and the chromatographic and mass spectrometric analysis results were evaluated. In general, matrix-assisted laser desorption/ionization (MALDI) time-of-flight MS of permethylated oligosaccharide mixtures carried out in six laboratories yielded good quantitation, and the results can be correlated to those of chromatography of reductive amination derivatives. For underivatized oligosaccharide alditols, graphitized carbon-liquid chromatography (LC)/electrospray ionization (ESI) MS detecting deprotonated molecules in the negative ion mode provided acceptable quantitation. The variance of the results among these three methods was small. Detailed analyses of tryptic glycopeptides employing either nano LC/ESI MS/MS or MALDI MS demonstrated excellent capability to determine site-specific or subclass-specific glycan profiles in these samples. Taking into account the variety of MS technologies and options for distinct protocols used in this study, the results of this multi-institutional study indicate that MS-based analysis appears as the efficient method for identification and quantitation of oligosaccharides in glycomic studies and endorse the power of MS for glycopeptide characterization with high sensitivity in proteomic programs.

The core fucosylation (␣1,6-fucosylation) of glycoproteins is widely distributed in mammalian tissues, and is altered under pathological conditions. To investigate physiological functions of the core fucose, we generated ␣1,6-fucosyltransferase (Fut8)-null mice and found that disruption of Fut8 induces severe growth retardation and death during postnatal development. Histopathological analysis revealed that Fut8 ؊/؊ mice showed emphysema-like changes in the lung, verified by a physiological compliance analysis. Biochemical studies indicated that lungs from Fut8 ؊/؊ mice exhibit a marked overexpression of matrix metalloproteinases (MMPs), such as MMP-12 and MMP-13, highly associated with lung-destructive phenotypes, and a down-regulation of extracellular matrix (ECM) proteins such as elastin, as well as retarded alveolar epithelia cell differentiation. These changes should be consistent with a deficiency in TGF-␤1 signaling, a pleiotropic factor that controls ECM homeostasis by down-regulating MMP expression and inducing ECM protein components. In fact, Fut8 ؊/؊ mice have a marked dysregulation of TGF-␤1 receptor activation and signaling, as assessed by TGF-␤1 binding assays and Smad2 phosphorylation analysis. We also show that these TGF-␤1 receptor defects found in Fut8 ؊/؊ cells can be rescued by reintroducing Fut8 into Fut8 ؊/؊ cells. Furthermore, exogenous TGF-␤1 potentially rescued emphysema-like phenotype and concomitantly reduced MMP expression in Fut8 ؊/؊ lung. We propose that the lack of core fucosylation of TGF-␤1 receptors is crucial for a developmental and progressive͞ destructive emphysema, suggesting that perturbation of this function could underlie certain cases of human emphysema.fucosylation ͉ glycobiology ͉ matrix metalloproteinase

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Although estrogen is known to activate endothelial nitric oxide synthase (eNOS) in the vascular endothelium, the molecular mechanism responsible for this effect remains to be elucidated. In studies of both human umbilical vein endothelial cells ( The inhibitory effect of estrogen on the development of atherosclerosis has been suggested by abundant human epidemiological and animal experimental data (1-9). The incidence of atherosclerotic diseases is lower in premenopausal women than in men, steeply rises in postmenopausal women, and is reduced to premenopausal levels in postmenopausal women who receive estrogen therapy (10 -12). Until recently, the atheroprotective effects of estrogen were attributed principally to the effects on serum lipid concentrations. However, estrogeninduced alterations in serum lipids account for only approximately one-third of the observed clinical benefits of estrogen (12)(13)(14). Recent evidence suggests that the direct actions of estrogen on blood vessels contribute to the cardioprotective effects of estrogen (13, 15). There are many kinds of direct effects of estrogen on blood vessels, such as estrogen-induced increases of vasodilatation and inhibition of the response of blood vessels to injury and the development of atherosclerosis. However, the molecular mechanism underlying the estrogeninduced vasodilatation has not yet been determined. Several studies suggest that a key mediator of this vasodilator response could be the endothelium-derived relaxing factor nitric oxide (NO), and that brief treatment with estrogen increases basal NO release in endothelial cells without elevation of eNOS mRNA or protein (16). Estrogen activates endothelial nitric oxide synthase (eNOS) without altering expression of the eNOS gene in vascular endothelium (17)(18)(19)(20). However, the details of the mechanism of the estrogen-induced eNOS activation are not yet well understood.The serine/threonine kinase termed Akt or protein kinase B (PKB) 1 is an important regulator of various cellular processes, including glucose metabolism and cell survival (21, 22). Activation of receptor tyrosine kinases and G-protein-coupled receptors, and stimulation of cells by mechanical force, can lead to the phosphorylation and activation of . Akt was identified as a downstream component of survival signaling through phosphatidylinositol 3-kinase (PI3K) (26 -30). Akt may be regulated by both phosphorylation and the direct binding of PI3K lipid products to the Akt pleckstrin homology domain. Akt can then phosphorylate substrates such as glycogen synthase kinase-3, 6-phosphofructo-2-kinase, and BAD. More recently, it was found that eNOS is also an Akt substrate and is activated by Akt-dependent phosphorylation to release NO in endothelial cells (31-34).The actions of estrogen can be mediated by the classical nuclear receptors, ER␣ and ER␤ (35,36) or through other putative membrane receptors. By definition, rapid effects of estrogen that involve nongenomic mechanisms are independent of transcriptional activation by the nuclea...