Glycosylation of interleukin-6 purified from normal human blood mononuclear cells.

Interleukin 6 (IL-6) is a glycosylated cytokine which is important in exerting cell-specific growth-inducing, growth-inhibiting and differentiation-inducing effects. IL-6 produced in mammalian cell lines is heterogeneous, reflecting specific cell-type-dependent post-translational modifications. Native IL-6 was purified from human blood mononuclear cells and the oligosaccharides released, radiolabelled and sequenced by a combination of sequential exoglycosidase digestion using Bio-Gel P-4 high-resolution gel chromatography and acetolysis. N- and O-linked glycans were found. The N-linked glycans were sialylated di- and tri-antennary complex-type and oligomannose-type structures. However, the most predominant N-linked oligosaccharide was a small tetrasaccharide with the sequence Man alpha 6Man beta 4GlcNAc beta 4GlcNAc. This is the first report of this structure on a circulating glycoprotein. This structure has only previously been reported to be present on the syncytiotrophoblast of human placenta. The presence of the oligomannose structures and the mannose-terminating tetrasaccharide on IL-6 may be important in maintaining a high local concentration of the cytokine while limiting its systemic serum level via interaction with soluble mannose-binding serum lectins.     

Comparative analysis of the N-glycans of rat, mouse and human Thy-1. Site-specific oligosaccharide patterns of neural Thy-1, a member of the immunoglobulin superfamily

Protein structure and tissue type are known to influence glycosylationof proteins. We have previously investigated the N-glycans ateach of the three glycosylation sites of the cell surface glycoproteinThy-1 when isolated from rat brain and thymocytes. Here we reporta comparative analysis of the site-specific N-glycosylationpatterns from rat (Asn 23, 74, 98), mouse (Asn 23, 75, 99) andhuman (Asn 23, 60, 100) neural Thy-1. Despite considerable differencesin amino acid sequence, the results show a remarkable conservationof the pattern of N-glycans at corresponding sites between thethree species, as judged by chromatographic comparisons andglycosidase susceptibility. This is particularly marked forsites at Asn 74/75 in rat/mouse and the equivalent site at 60in human Thy-1, as well as for sites at Asn 98/99 and 100, respectively.The sites at Asn 23 in rat/mouse also contained almost identicalglycosylation paterns, but at this site human Thy-1 showed significantlydifferent glycosylation patterns. These site glycosylation patternsare discussed in relation to the likely accessibility of theoligosaccharides for processing. It is known that within a species,the glycosylation of Thy-1 is tissue specific; therefore, thisdegree of conservation of glycosylation of Thy-1 expressed inthe same tissue in different species is all the more striking,given the known variation between species in the amino acidsequence of Thy-1. It is therefore proposed that neural cellshave a particular requirement for specific surface carbohydratesand that the Thy-1 polypep-tide serves as an appropriate carrierfor these structures. glycosylation site-specific Thy-1     

Quantitative and qualitative differences in the in vivo response of NKT cells to distinct alpha- and beta-anomeric glycolipids

NKT cells represent a unique subset of immunoregulatory T cells that recognize glycolipid Ags presented by the MHC class I-like molecule CD1d. Because of their immunoregulatory properties, NKT cells are attractive targets for the development of immunotherapies. The prototypical NKT cell ligand alpha-galactosylceramide (alpha-GalCer), originally isolated from a marine sponge, has potent immunomodulatory activities in mice, demonstrating therapeutic efficacy against metastatic tumors, infections, and autoimmune diseases, but also has a number of adverse side effects. In vivo administration of alpha-GalCer to mice results in the rapid activation of NKT cells, which is characterized by cytokine secretion, surface receptor down-regulation, expansion, and secondary activation of a variety of innate and adaptive immune system cells. In this study, we have evaluated the in vivo immune response of mice to a set of structural analogues of alpha-GalCer. Our results show that, contrary to current thinking, beta-anomeric GalCer can induce CD1d-dependent biological activities in mice, albeit at lower potency than alpha-anomeric GalCer. In addition, we show that the response of NKT cells to distinct GalCer differs not only quantitatively, but also qualitatively. These findings indicate that NKT cells can fine-tune their immune responses to distinct glycolipid Ags in vivo, a property that may be exploited for the development of effective and safe NKT cell-based immunotherapies.     

Protein kinase B, P34cdc2 kinase, and p21 ras GTP-binding in kidneys of aging rats

Renal nephropathy present in male Wistar rats more than 13 months of age was reported as an indication that the rats were in renal failure. In this study, the renal tissue damage at 14 months of age in male Munich Wistar rats was similar to that reported for Wistar rats, indicating that Munich Wistar rats could be another model for study of kidney function in the aging rat. The usual renal response to injury involves increased cell division and/or reparative processes that involve tyrosine kinase activity (TyrK) and/or guanosine triphosphate-binding (G) protein signal trans-duction pathways. This study reveals the presence of renal tissue damage coinciding with significantly reduced activity of Ras, Akt, and p34cdc2 kinase, the signaling proteins that regulate cell division and/or growth, in renal cortical tissues of aging rats compared to young rats (P < 0.005, P < 0.005, and P< 0.001, respectively). These results suggest that proteins involved in signal transduction pathways associated with cell replication are downregulated in the aging kidney cortex at a time when renal cellular damage is also present.     

Energized mitochondria increase the dynamic range over which inositol 1,4,5-trisphosphate activates store-operated calcium influx

In eukaryotic cells, activation of cell surface receptors that couple to the phosphoinositide pathway evokes a biphasic increase in intracellular free Ca2+ concentration: an initial transient phase reflecting Ca2+ release from intracellular stores, followed by a plateau phase due to Ca2+ influx. A major component of this Ca2+ influx is store-dependent and often can be measured directly as the Ca2+ release-activated Ca2+ current (I(CRAC)). Under physiological conditions of weak intracellular Ca2+ buffering, respiring mitochondria play a central role in store-operated Ca2+ influx. They determine whether macroscopic I(CRAC) activates or not, to what extent and for how long. Here we describe an additional role for energized mitochondria: they reduce the amount of inositol 1,4,5-trisphosphate (InsP3) that is required to activate I(CRAC). By increasing the sensitivity of store-operated influx to InsP3, respiring mitochondria will determine whether modest levels of stimulation are capable of evoking Ca2+ entry or not. Mitochondrial Ca2+ buffering therefore increases the dynamic range of concentrations over which the InsP3 is able to function as the physiological messenger that triggers the activation of store-operated Ca2+ influx.