The pathway by which Semliki Forest virus (SFV), a membrane-containing animal virus, enters BHK-21 cells was studied morphologically and biochemically . After attaching to the cell surface, the majority of viruses was rapidly trapped into coated pits, internalized by endocytosis in coated vesicles, and sequestered into intracellular vacuoles and lysosomes . Direct penetration of viruses through the plasma membrane was never observed .To assess the possible involvement of lysosomes in the release of the genome into the cytoplasm, the effect of five lysosomotropic agents, known to increase the lysosomal pH, was tested. All of these agents inhibited SFV infectivity and one, chloroquine (the agent studied in most detail), inhibited a very early step in the infection but had no effect on binding, endocytosis, or intracellular distribution of SFV . Thus, the inhibitory effect was concluded to be either on penetration of the nucleocapsid into the cytoplasm or on uncoating of the viral RNA .Possible mechanisms for the penetration of the genome into the cytoplasm were studied in vitro, using phospholipid-cholesterol liposomes and isolated SFV . When the pH was 6 .0 or lower, efficient fusion of the viral membranes and the liposomal membranes occurred, resulting in the transfer of the nucleocapsid into the liposomes . Infection of cells could also be induced by brief low pH treatment of cells with bound SFV under conditions where the normal infection route was blocked .The results suggest that the penetration of the viral genome into the cytosol takes place intracellularly through fusion between the limiting membrane of intracellular vacuoles and the membrane of viruses contained within them . The low pH required for fusion together with the inhibitory effect of lysosomotropic agents implicate lysosomes, or other intracellular vacuoles with sufficiently low pH, as the main sites of penetration . KEY WORDS coated vesicleslysosomes " from the extracellular space to the cytosol . This endocytosis -membrane fusion process requires transport through one or more lysosomotropic agents membrane barriers . For animal viruses the mechanisms involved are poorly understood . Data from To infect a cell, a virus must transfer its genome electron microscopy suggest that penetration oc-404 J . CELL BIOLOGY

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The high molecular mass glycosaminoglycan hyaluronan (HA) can become modified by the covalent attachment of heavy chains (HCs) derived from the serum protein inter-alpha-inhibitor (IalphaI), which is composed of three subunits (HC1, HC2 and bikunin) linked together via a chondroitin sulfate moiety. The formation of HC.HA is likely to play an important role in the stabilization of HA-rich extracellular matrices in the context of inflammatory disease (e.g. arthritis) and ovulation. Here, we have characterized the complexes formed in vitro between purified human IalphaI and recombinant human TSG-6 (an inflammation-associated protein implicated previously in this process) and show that these complexes (i.e. TSG-6 x HC1 and TSG-6 x HC2) act as intermediates in the formation of HC x HA. This is likely to involve two transesterification reactions in which an ester bond linking an HC to chondroitin sulfate in intact IalphaI is transferred first onto TSG-6 and then onto HA. The formation of TSG-6 x HC1 and TSG-6 x C2 complexes was accompanied by the production of bikunin x HC2 and bikunin x HC1 by-products, respectively, which were observed to break down, releasing free bikunin and HCs. Both TSG-6 x HC formation and the subsequent HC transfer are metal ion-dependent processes; these reactions have a requirement for either Mg2+ or Mn2+ and are inhibited by Co2+. TSG-6, which is released upon the transfer of HCs from TSG-6 onto HA, was shown to combine with IalphaI to form new TSG-6 x HC complexes and thus be recycled. The finding that TSG-6 acts as cofactor and catalyst in the production of HC x HA complexes has important implications for our understanding of inflammatory and inflammation-like processes.

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TSG-6, the secreted product of tumor necrosis factorstimulated gene-6, is not constitutively expressed but is up-regulated in various cell-types during inflammatory and inflammation-like processes. The mature protein is comprised largely of contiguous Link and CUB modules, the former binding several matrix components such as hyaluronan (HA) and aggrecan. Here we show that this domain can also associate with the glycosaminoglycan heparin/heparan sulfate. Docking predictions and sitedirected mutagenesis demonstrate that this occurs at a site distinct from the HA binding surface and is likely to involve extensive electrostatic contacts. Despite these glycosaminoglycans binding to non-overlapping sites on the Link module, the interaction of heparin can inhibit subsequent binding to HA, and it is possible that this occurs via an allosteric mechanism. We also show that heparin can modify another property of the Link module, i.e. its potentiation of the anti-plasmin activity of inter-␣-inhibitor (I␣I). Experiments using the purified components of I␣I indicate that TSG-6 only binds to the bikunin chain and that this is at a site on the Link module that overlaps the HA binding surface. The association of heparin with the Link module significantly increases the anti-plasmin activity of the TSG-6⅐I␣I complex. Changes in plasmin activity have been observed previously at sites of TSG-6 expression, and the results presented here suggest that TSG-6 is likely to contribute to matrix remodeling, at least in part, through down-regulation of the protease network, especially in locations containing heparin/heparan sulfate proteoglycans. The differential effects of HA and heparin on TSG-6 function provide a mechanism for its regulation and functional partitioning in particular tissue microenvironments.TSG-6 1 (the secreted product of tumor necrosis factor-stimulated gene-6), a protein composed mainly of contiguous Link and CUB modules, is not constitutively expressed in adult tissues but is up-regulated in many different inflammatory diseases that often involve remodeling of the extracellular matrix (ECM) (1). These include rheumatoid arthritis and osteoarthritis (2, 3), Kawasaki disease (4), systemic lupus erythematosus (5), and asthma. 2 TSG-6 is also expressed in normal physiological processes involving ECM reorganization, notably following blood vessel wall injury (6), during ovulation (6 -10), and in cervical ripening (11). TSG-6 binds to several components of the ECM through its Link module domain; i.e. the glycosaminoglycans (GAGs) hyaluronan (HA) (12-17) and chondroitin-4-sulfate (13), as well as the G1 domain of aggrecan (18) and pentraxin-3 (19, 20). Interactions between TSG-6 and the serine protease inhibitor inter-␣-inhibitor (I␣I) have also been described (21-27). I␣I is a proteoglycan and consists of three polypeptides (heavy chain 1 (HC1), heavy chain 2 (HC2) and bikunin), covalently linked by a chondroitin sulfate moiety, which originates from Ser-10 of bikunin (28). There is evidence that TSG-6 mediates the cross-li...

TSG-6 protein (the secreted product of the tumor necrosis factor-stimulated gene-6), a hyaluronan-binding protein comprised mainly of a Link and CUB module arranged in a contiguous fashion, has been shown previously to be a potent inhibitor of neutrophil migration in an in vivo model of acute inflammation (Wisniewski, H. G., Hua, J. C., Poppers, D. M., Naime, D., Vilcek, J., and Cronstein, B. N. (1996) J. Immunol. 156, 1609 -1615). It was hypothesized that this activity of TSG-6 was likely to be mediated by its potentiation of inter-␣-inhibitor anti-plasmin activity (causing a down-regulation of the protease network), which was reliant on these proteins forming a stable, probably covalent ϳ120-kDa complex. Here we have shown that the recombinant Link module from human TSG-6 (Link_TSG6; expressed in Escherichia coli) has an inhibitory effect on neutrophil influx into zymosan A-stimulated murine air pouches, equivalent to that of full-length protein (which we produced in a Drosophila expression system). The active dose of 1 g of Link_TSG6 per mouse (administered intravenously) also resulted in a significant reduction in the concentrations of various inflammatory mediators (i.e. tumor necrosis factor-␣, KC, and prostaglandin E 2 ) in air pouch exudates. Link_TSG6, although unable to form a stable complex with inter-␣-inhibitor (under conditions that promote maximum complex formation with the fulllength protein), could potentiate its anti-plasmin activity. This demonstrates that formation of an ϳ120-kDa TSG-6⅐inter-␣-inhibitor complex is not required for TSG-6 to enhance the serine protease inhibitory activity of inter-␣-inhibitor. Six single-site Link_TSG6 mutants (with wild-type folds) were compared for their abilities to inhibit neutrophil migration in vivo, bind hyaluronan, and potentiate inter-␣-inhibitor. These experiments indicate that all of the inhibitory activity of TSG-6 resides within the Link module domain, and that this anti-inflammatory property is not related to either its hyaluronan binding function or its potentiation of the anti-plasmin activity of inter-␣-inhibitor.

We describe a cell-free system in which the membrane glycoprotein of vesicular stomatitis virus is rapidly and efficiently transported to membranes of the Golgi complex by a process resembling intracellular protein transport. Transport in vitro is energy dependent and is accompanied by terminal glycosylation of the membrane glycoprotein (dependent upon UDP-lcNAc and resulting in resistance to endo-f-Nacetylglucosaminidase H). The elucidation of the mechanisms of assembly of cellular membranes and of the organelles that these membranes define poses a major challenge to cell biologists. In particular, it will be important to understand how distinct sets of proteins are delivered to the different subcellular organelles so as to confer upon them their distinct functions. How is this highly specific intracellular transport and sorting of proteins accomplished?An essential step towards obtaining a molecular description of these complex cellular events will be to achieve conditions under which the same transport of nascent proteins can proceed in cell-free extracts, for only then can the tools of biochemistry be fruitfully applied. We describe here experiments demonstrating that it is possible to obtain transfer of protein between specific organelles in a cell-free extract by a reaction resembling that of intracellular protein transport. For these studies we have utilized Chinese hamster ovary (CHO) cells infected with vesicular stomatitis virus (VSV) as a well-defined system in which to investigate the transport of a membrane protein destined for the plasma membrane (1).The viral glycoprotein (G protein) that will reside in the membrane of the mature virion is the only glycoprotein synthesized in VSV-infected cells. Like cellular surface glycoproteins, G is synthesized in the endoplasmic reticulum (ER), is transported to the Golgi complex,* and is then transported to the plasma membrane (1). Clathrin-coated vesicles appear to mediate both of these transport steps (6). The small genome of VSV (encoding only five proteins, all found in virions) provides assurance that the maturation of G follows host-specific pathways.During its synthesis in the ER, G acquires two mannose-rich oligosaccharides that are subsequently processed as G passes through the Golgi complex (2,3,7). This change in oligosaccharide structure provides an indirect means for assaying the arrival of G at the Golgi complex. Endo-H has proved useful in this regard, because this enzyme will cleave the mannose-rich precursor oligosaccharides of G, causing a marked reduction of apparent molecular weight, but will not attack the Golgiprocessed oligosaccharides (7).The general approach we have taken to obtain transport of

Vertebrates produce various chondroitin sulfate proteoglycans (CSPGs) that are important structural components of cartilage and other connective tissues. CSPGs also contribute to the regulation of more specialized processes such as neurogenesis and angiogenesis. Although many aspects of CSPGs have been studied extensively, little is known of where the CS chains are attached on the core proteins and so far, only a limited number of CSPGs have been identified. Obtaining global information on glycan structures and attachment sites would contribute to our understanding of the complex proteoglycan structures and may also assist in assigning CSPG specific functions. In the present work, we have developed a glycoproteomics approach that characterizes CS linkage regions, attachment sites, and identities of core proteins. CSPGs were enriched from human urine and cerebrospinal fluid samples by strong-anion-exchange chromatography, digested with chondroitinase ABC, a specific CSlyase used to reduce the CS chain lengths and subsequently analyzed by nLC-MS/MS with a novel glycopeptide search algorithm. The protocol enabled the identification of 13 novel CSPGs, in addition to 13 previously established CSPGs, demonstrating that this approach can be routinely used to characterize CSPGs in complex human samples. Surprisingly, five of the identified CSPGs are traditionally defined as prohormones (cholecystokinin, chromogranin A, neuropeptide W, secretogranin-1, and secretogranin-3), typically stored and secreted from granules of endocrine cells. We hypothesized that the CS side chain may influence the assembly and structural organization of secretory granules and applied surface plasmon resonance spectroscopy to show that CS actually promotes the assembly of chromogranin A core proteins in vitro. This activity required mild acidic pH and suggests that the CS-side chains may also influence the self-assembly of chromogranin A in vivo giving a possible explanation to previous observations that chromogranin A has an inherent property to assemble in the acidic milieu of secretory granules. Molecular & Cellular