Previous studies have indicated that endotoxin and other bacterial and protozoal products can stimulate macrophages to produce a factor that can suppress the activity of the enzyme lipoprotein lipase (LPL), in vivo and in vitro. In the present report we describe the purification of this factor, cachectin, to apparent homogeneity from the conditioned medium of endotoxin-stimulated RAW 264.7 cells. The isolated protein has an isoelectric point of 4.7 and a subunit molecular weight of 17,000. Although cachectin's isoelectric point and molecular weight are similar to those described for interleukin 1, pure cachectin has no leukocyte-activating factor (LAF) activity. Cachectin at a concentration of 10(-11) M has the ability to suppress the LPL activity of the 3T3-L1 adipocyte cell line by 80%. Binding studies using radio-labeled cachectin and 3T3-L1 adipocytes and C2 myotubules revealed approximately 10(4) high-affinity receptors per cell on both cell types (Ka, 3 X 10(9]. Cachectin receptors were also present on liver membranes but were absent on erythrocytes and lymphocytes. The isolation of cachectin and characterization of its receptor should facilitate further investigations into the role of cachectin and other macrophage mediators in the metabolic derangements that occur during infection and cachexia.
The presence of a distinct nucleus, the compartment for confining the genome, transcription and RNA maturation, is a central (and eponymous) feature that distinguishes eukaryotes from prokaryotes. Structural integrity of the nucleus is maintained by the nuclear envelope (NE). A crucial element of this structure is the nuclear pore complex (NPC), a macromolecular machine with over 90 protein components, which mediates nucleo-cytoplasmic communication. We investigated the provenance of the conserved domains found in these perinuclear proteins and reconstructed a parsimonious scenario for NE and NPC evolution by means of comparative-genomic analysis of their components from the available sequences of 28 sequenced eukaryotic genomes. We show that the NE and NPC proteins were tinkered together from diverse domains, which evolved from prokaryotic precursors at different points in eukaryotic evolution, divergence from pre-existing eukaryotic paralogs performing other functions, and de novo. It is shown that several central components of the NPC, in particular, the RanGDP import factor NTF2, the HEH domain of Src1p-Man1, and, probably, also the key domains of karyopherins and nucleoporins, the HEAT/ARM and WD40 repeats, have a bacterial, most likely, endosymbiotic origin. The specialized immunoglobulin (Ig) domain in the globular tail of the animal lamins, and the Ig domains in the nuclear membrane protein GP210 are shown to be related to distinct prokaryotic families of Ig domains. This suggests that independent, late horizontal gene transfer events from bacterial sources might have contributed to the evolution of perinuclear proteins in some of the major eukaryotic lineages. Snurportin 1, one of the highly conserved karyopherins, contains a cap-binding domain which is shown to be an inactive paralog of the guanylyl transferase domain of the mRNA-capping enzyme, exemplifying recruitment of paralogs of pre-exsiting proteins for perinuclear functions. It is shown that several NPC proteins containing super-structure- forming alpha-helical and beta-propeller modules are most closely related to corresponding proteins in the cytoplasmic vesicle biogenesis and coating complexes. From these observations, we infer an autogenous scenario of nuclear evolution in which the nucleus emerged in the primitive eukaryotic ancestor (the "prekaryote") as part of cell compartmentalization triggered by archaeo-bacterial symbiosis. A pivotal event in this process was the radiation of Ras-superfamily GTPases yielding Ran, the key regulator of nuclear transport. A primitive NPC with approximately 20 proteins and a Src1p-Man1-like membrane protein with a DNA-tethering HEH domain are inferred to have been integral perinuclear components in the las common ancestor of modern eukaryotes.
-O-linked N-acetylglucosamine (O-GlcNAc) is an abundant and dynamic post-translational modification implicated in protein regulation that appears to be functionally more similar to phosphorylation than to classical glycosylation. There are nucleocytoplasmic enzymes for the attachment and removal of O-GlcNAc. Here, we further characterize the recently cloned -N-acetylglucosaminidase, O-GlcNAcase. Both recombinant and purified endogenous O-GlcNAcase rapidly release free GlcNAc from O-GlcNAc-modified peptide substrates. The recombinant enzyme functions as a monomer and has kinetic parameters (K m ؍ 1.1 mM for paranitrophenyl-GlcNAc, k cat ؍ 1 s ؊1 ) that are similar to those of lysosomal hexosaminidases. The endogenous O-GlcNAcase appears to be in a complex with other proteins and is predominantly localized to the cytosol. Overexpression of the enzyme in living cells results in decreased O-GlcNAc modification of nucleocytoplasmic proteins. Finally, we show that the enzyme is a substrate for caspase-3 but, suprisingly, the cleavage has no effect on in vitro O-GlcNAcase activity. These studies support the identification of this protein as an O-GlcNAcase and identify important interactions and modifications that may regulate the enzyme and O-GlcNAc cycling.Multiple nucleocytoplasmic proteins, including transcription factors, cytoskeletal proteins, oncogenes, and kinases, are posttranslationally modified with -N-acetylglucosamine (O-GlcNAc) 1 (1-3). In each case studied, the glycan has a turnover rate that is much faster than that of the protein to which it is attached (4, 5). The O-GlcNAc modification of certain proteins is modulated by various extracellular stimuli and growth conditions (6, 7). Thus, based on its dynamic and inducible nature, O-GlcNAc is most likely a regulatory modification, more similar to phosphorylation than to classical glycosylation (8). In further support of this hypothesis, there are nucleocytoplasmic enzymes for the attachment (O-GlcNAc transferase, OGT) and for the removal (O-GlcNAcase) of O-GlcNAc, analogous to the kinases and phosphatases for phosphate.The protein uridine diphospho-N-acetylglucosamine: polypeptide -N-acetylglucosaminyltransferase (OGT) has been cloned and characterized in our laboratory and others (10, 11). OGT is highly conserved from Caenorhabditis elegans to humans and is essential for mouse embryonic stem cell viability (12). OGT contains multiple tetratricopeptide repeats that appear to mediate the enzyme's trimerization, substrate recognition, and protein-protein interactions (13,14). Furthermore, the enzyme is modified by tyrosine phosphorylation, as well as O-GlcNAc (10). A variety of splice variants of this enzyme appear in the National Center for Biotechnology Information data base, but they have not been examined. The enzyme is also exquisitely responsive to the concentration of UDP-GlcNAc, the donor sugar nucleotide (13). This multitude of protein-binding domains, post-translational modifications, splice variants, and substrate sensitivity could allow ...
A soluble form of the mannose receptor (sMR) has been found in conditioned medium of primary macrophages in vitro and in mouse serum. sMR was released as a single species, had a smaller size than the cellassociated form, and accumulated in macrophage-conditioned medium, in a cytokine-regulated manner, to levels comparable with those found for cell-associated mannose receptor. Pulse-chase experiments showed that sMR production in culture occurred by constitutive cleavage of pre-existing full-length protein. A binding assay was developed to determine the sugar specificity of sMR and its ability to interact with pathogens and particulate antigens (i.e. Candida albicans and zymosan). Protease inhibitor studies suggested that sMR was produced by cleavage of an intact mannose receptor by a matrix metalloprotease or ADAM metalloprotease. A role for sMR in the immune response is proposed based on its binding properties, regulation by cytokines, and the previous discovery of putative ligands for the cysteine-rich domain of the mannose receptor in lymph nodes and spleen. The mannose receptor (MR)1 is a type I integral membrane glycoprotein expressed on many macrophage (MØ) subtypes, monocyte-derived dendritic cells, and hepatic endothelium (1-4). MR is the founding member of a family of molecules sharing the same basic domain structure: an NH 2 -terminal cysteinerich domain; a fibronectin type II domain; and a variable number of C-type lectin carbohydrate recognition domains (CRDs), eight in the case of MR, followed by a transmembrane domain and a COOH-terminal intracellular domain (5-7). The other members of the family are: (i) the phospholipase A 2 receptor, which contains eight CRDs and is widely distributed (8 -10); (ii) the receptor DEC-205, which contains 10 CRDs and is expressed mostly by dendritic cells in T-cell areas of secondary lymphoid organs and by thymic and intestinal epithelia (11-13); and (iii) a recently described molecule found by its homology to the C-type lectin domain of E-selectin (14).Mannosylated molecules and particles can be endocytosed and phagocytosed through their interaction with the MR (3, 4, 15-19). The domains likely to be involved in this process are the CRDs and the transmembrane and the COOH-terminal intracellular domains. Neither the cysteine-rich domain nor the fibronectin type II domain seems to be required (18,20). MR synthesis and function are regulated by MØ maturation, lipopolysaccharide (LPS) (3), and cytokines; inhibited by interferon (IFN)-␥ (6); and up-regulated by 22).Using as a probe a chimeric protein containing the cysteinerich domain of the MR fused to the Fc region of human IgG1 (CR-Fc), we found putative ligand(s) for this domain of the MR in marginal zone metallophilic and subcapsular sinus MØ in naive mice and in the germinal centers of immunized mice. CR-Fc ϩ cells with dendritic morphology migrated toward the follicular areas and paracortex of lymph nodes during a secondary immune response (23). We hypothesized a role for a soluble form of the MR in the transport...
Transverse myelitis (TM) is an idiopathic inflammatory disorder of the spinal cord. The authors observed cases of recurrent TM in patients where anti-Ro (SSA) antibodies were present and therefore performed a case-control study to examine the frequency of anti-Ro autoantibodies in patients with recurrent TM and control subjects. Antibodies to 52-kd Ro were demonstrated in 77% of cases (10/13) compared with only 33% of control subjects (4/12).
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