Emerging roles of protein mannosylation in inflammation and infection.

Proteins are frequently modified by complex carbohydrates (glycans) that play central roles in maintaining the structural and functional integrity of cells and tissues in humans and lower organisms. Mannose forms an essential building block of protein glycosylation, and its functional involvement as components of larger and diverse α-mannosidic glycoepitopes in important intra- and intercellular glycoimmunological processes is gaining recognition. With a focus on the mannose-rich asparagine (N-linked) glycosylation type, this review summarises the increasing volume of literature covering human and non-human protein mannosylation, including their structures, biosynthesis and spatiotemporal expression. The review also covers their known interactions with specialised host and microbial mannose-recognising C-type lectin receptors (mrCLRs) and antibodies (mrAbs) during inflammation and pathogen infection. Advances in molecular mapping technologies have recently revealed novel immuno-centric mannose-terminating truncated N-glycans, termed paucimannosylation, on human proteins. The cellular presentation of α-mannosidic glycoepitopes on N-glycoproteins appears tightly regulated; α-mannose determinants are relative rare glycoepitopes in physiological extracellular environments, but may be actively secreted or leaked from cells to transmit potent signals when required. Simultaneously, our understanding of the molecular basis on the recognition of mannosidic epitopes by mrCLRs including DC-SIGN, mannose receptor, mannose binding lectin and mrAb is rapidly advancing, together with the functional implications of these interactions in facilitating an effective immune response during physiological and pathophysiological conditions. Ultimately, deciphering these complex mannose-based receptor-ligand interactions at the detailed molecular level will significantly advance our understanding of immunological disorders and infectious diseases, promoting the development of future therapeutics to improve patient clinical outcomes.

Mannose-capped lipoarabinomannan from Mycobacterium tuberculosis preferentially inhibits sphingosine-1-phosphate-induced migration of Th1 cells.

Chemokine receptor cross-desensitization provides an important mechanism to regulate immune cell recruitment at sites of inflammation. We previously reported that the mycobacterial cell wall glycophospholipid mannose-capped lipoarabinomannan (ManLAM) could induce human peripheral blood T cell chemotaxis. Therefore, we examined the ability of ManLAM to desensitize T cells to other chemoattractants as a potential mechanism for impaired T cell homing and delayed lung recruitment during mycobacterial infection. We found that ManLAM pretreatment inhibited in vitro migration of naive human or mouse T cells to the lymph node egress signal sphingosine-1-phosphate (S1P). Intratracheal administration of ManLAM in mice resulted in significant increases in T cells, primarily CCR5(+) (Th1) cells, in lung-draining lymph nodes. To investigate the selective CCR5 effect, mouse T cells were differentiated into Th1 or Th2 populations in vitro, and their ability to migrate to S1P with or without ManLAM pretreatment was analyzed. ManLAM pretreatment of Th1 populations inhibited S1P-induced migration but had no effect on Th2 cell S1P-directed migration, suggesting a differential effect by S1P on the two subsets. The PI3K/AKT inhibitor Ly294002 inhibited S1P-directed migration by Th1 cells, whereas the ERK inhibitor U0126 inhibited Th2 cell S1P-directed migration. These observations demonstrate that S1P-induced migratory responses in Th1 and Th2 lymphocytes occurs via different signaling pathways and suggests further that the production of ManLAM during Mycobacterium tuberculosis infection may function to sequester Th1 cells in lung-draining lymph nodes, thereby delaying their recruitment to the lung.

Cyanovirin-N inhibits mannose-dependent Mycobacterium-C-type lectin interactions but does not protect against murine tuberculosis.

Cyanovirin-N (CV-N) is a mannose-binding lectin that inhibits HIV-1 infection by blocking mannose-dependent target cell entry via C-type lectins. Like HIV-1, Mycobacterium tuberculosis expresses mannosylated surface structures and exploits C-type lectins to gain cell access. In this study, we investigated whether CV-N, like HIV-1, can inhibit M. tuberculosis infection. We found that CV-N specifically interacted with mycobacteria by binding to the mannose-capped lipoglycan lipoarabinomannan. Furthermore, CV-N competed with the C-type lectins DC-SIGN and mannose receptor for ligand binding and inhibited the binding of M. tuberculosis to dendritic cells but, unexpectedly, not to macrophages. Subsequent in vivo infection experiments in a mouse model demonstrated that, despite its activity, CV-N did not inhibit or delay M. tuberculosis infection. This outcome argues against a critical role for mannose-dependent C-type lectin interactions during the initial stages of murine M. tuberculosis infection and suggests that, depending on the circumstances, M. tuberculosis can productively infect cells using different modes of entry.

Role of endothelial N-glycan mannose residues in monocyte recruitment during atherogenesis.

OBJECTIVE: Upregulated expression of endothelial adhesion molecules and subsequent binding to cognate monocytic receptors are established paradigms in atherosclerosis. However, these proteins are the scaffolds, with their posttranslational modification with sugars providing the actual ligands. We recently showed that tumor necrosis factor-α increased hypoglycosylated (mannose-rich) N-glycans on the endothelial surface. In the present study, our aim was to determine whether (1) hypoglycosylated N-glycans are upregulated by proatherogenic stimuli (oscillatory flow) in vitro and in vivo, and (2) mannose residues on hypoglycosylated endothelial N-glycans mediate monocyte rolling and adhesion. METHODS AND RESULTS: Staining with the mannose-specific lectins concanavalin A and lens culinaris agglutinin was increased in human aortic endothelial cells exposed to oscillatory shear or tumor necrosis factor-α and at sites of plaque development and progression in both mice and human vessels. Increasing surface N-linked mannose by inhibiting N-glycan processing potentiated monocyte adhesion under flow during tumor necrosis factor-α stimulation. Conversely, enzymatic removal of high-mannose N-glycans, or masking mannose residues with lectins, significantly decreased monocyte adhesion under flow. These effects occurred without altering induced expression of adhesion molecule proteins. CONCLUSIONS: Hypoglycosylated (high mannose) N-glycans are present on the endothelial cell surface at sites of early human lesion development and are novel effectors of monocyte adhesion during atherogenesis.

Effect of N-acetyl-D-glucosamine on bovine sperm-oocyte interactions.

N-Acetyl-D-glucosamine (GlcNAc) is a major component of glycosaminoglycan, which is involved in sperm-oocyte interactions. We examined the effect of adding GlcNAc and other monosaccharides, D-mannose and D-fucose, to the in vitro fertilization (IVF) medium on bovine sperm-oocyte interactions. In medium in which sperm and a zona pellucida (ZP) were co-incubated with monosaccharides for 5 min, addition of GlcNAc (5 or 25 mM) significantly reduced the number of sperm that attached to the ZP. Pretreatment of gametes with GlcNAc (5 mM) prior to co-incubation also suppressed sperm-ZP attachment. Addition of GlcNAc (5 or 25 mM) to the medium in which sperm and a ZP were co-incubated for 5 h, however, significantly increased the number of sperm binding to and penetrating the ZP in a concentration-related manner. The other monosaccharides, D-fucose and D-mannose, did not have this effect. Supplementation of the sperm-oocyte co-incubation medium with 5 mM GlcNAc also enhanced the rate of polyspermic fertilization. When the ZPs were removed from the oocytes, GlcNAc did not affect the fertilization rate. Furthermore, incubation of sperm with 5 mM GlcNAc induced sperm membrane destabilization and an acrosome reaction, as evidenced by the hypo-osmotic swelling test and fluorescein isothiocyanate-labeled peanut agglutinin/propidium iodide (FITC-PNA/PI) staining. Finally, GlcNAc suppressed ZP hardening following fertilization, as determined by measuring the time required for pronase to dissolve the ZP. In conclusion, supplementation of IVF medium with GlcNAc has various effects on sperm-oocyte interactions including suppression of initial attachment, induction of sperm membrane destabilization and acrosome reaction, increase in the number of sperm secondarily bound to and penetrating the ZP, suppression of ZP hardening following sperm-oocyte co-incubation and increase in the rate of polyspermic fertilization.

Mannose-capped lipoarabinomannan- and prostaglandin E2-dependent expansion of regulatory T cells in human Mycobacterium tuberculosis infection.

We evaluated the role of regulatory T cells (CD4(+) CD25(+) Foxp3(+) cells, Tregs) in human Mycobacterium tuberculosis infection. Tregs were expanded in response to M. tuberculosis in healthy tuberculin reactors, but not in tuberculin-negative individuals. The M. tuberculosis mannose-capped lipoarabinomannan (ManLAM) resulted in regulatory T cell expansion, whereas the M. tuberculosis 19-kDa protein and heat shock protein 65 had no effect. Anti-IL-10 and anti-TGF-beta alone or in combination, did not reduce expansion of Tregs. In contrast, the cyclooxygenase enzyme-2 inhibitor NS398 significantly inhibited expansion of Tregs, indicating that prostaglandin E2 (PGE2) contributes to Treg expansion. Monocytes produced PGE2 upon culturing with heat-killed M. tuberculosis or ManLAM, and T cells from healthy tuberculin reactors enhanced PGE2 production by monocytes. Expanded Tregs produced significant amounts of TGF-beta and IL-10 and depletion of Tregs from PBMC of these individuals increased the frequency of M. tuberculosis-responsive CD4(+) IFN-gamma cells. Culturing M. tuberculosis-expanded Tregs with autologous CD8(+) cells decreased the frequency of IFN-gamma(+)cells. Freshly isolated PBMC from tuberculosis patients had increased percentages of Tregs, compared to healthy tuberculin reactors. These findings demonstrate that Tregs expand in response to M. tuberculosis through mechanisms that depend on ManLAM and PGE2.

The mannose cap of mycobacterial lipoarabinomannan does not dominate the Mycobacterium-host interaction.

Pathogenic mycobacteria have the ability to persist in phagocytic cells and to suppress the immune system. The glycolipid lipoarabinomannan (LAM), in particular its mannose cap, has been shown to inhibit phagolysosome fusion and to induce immunosuppressive IL-10 production via interaction with the mannose receptor or DC-SIGN. Hence, the current paradigm is that the mannose cap of LAM is a crucial factor in mycobacterial virulence. However, the above studies were performed with purified LAM, never with live bacteria. Here we evaluate the biological properties of capless mutants of Mycobacterium marinum and M. bovis BCG, made by inactivating homologues of Rv1635c. We show that its gene product is an undecaprenyl phosphomannose-dependent mannosyltransferase. Compared with parent strain, capless M. marinum induced slightly less uptake by and slightly more phagolysosome fusion in infected macrophages but this did not lead to decreased survival of the bacteria in vitro, nor in vivo in zebra fish. Loss of caps in M. bovis BCG resulted in a sometimes decreased binding to human dendritic cells or DC-SIGN-transfected Raji cells, but no differences in IL-10 induction were observed. In mice, capless M. bovis BCG did not survive less well in lung, spleen or liver and induced a similar cytokine profile. Our data contradict the current paradigm and demonstrate that mannose-capped LAM does not dominate the Mycobacterium-host interaction.

Inhibitory effects of d-mannose on trypanosomatid lysis induced by Serratia marcescens.

Studies were carried out on the effects of different carbohydrates on the lysis of Trypanosoma cruzi, Trypanosoma rangeli and erythocytes caused by the bacteria Serratia marcescens variants SM 365 and RPH. High concentrations of d-mannose were found to protect T. cruzi and T. rangeli markedly diminishing the lysis caused by S. marcescens. However, this carbohydrate is unable to interfere with the hemolysis induced by SM 365 and RPH variants. These results showed that the trypanolytic effect induced by S. marcescens SM 365 and RPH variants is dependent on d-mannose and distinct from the hemolytic activity, strongly suggesting that bacterial fimbriae are relevant to S. marcescens in lysis of parasites.

Lectin/glycan interactions play a role in recognition in a coral/dinoflagellate symbiosis.

Recognition is an important stage in the establishment of highly specific mutualistic associations. Yet, for the majority of symbioses, very few of the mechanisms involved in recognition and specificity are known. In this study, we provide evidence for a recognition mechanism at the onset of symbiosis between larvae of the coral Fungia scutaria and their endosymbiotic dinoflagellate algae. This recognition step occurs during initial cellular contact between the symbiotic partners through a lectin/glycan interaction. We determined that an intact algal cell surface was required for successful infection of F. scutaria larvae. Modification of the algal cell surface by enzymatic digestion with trypsin or N-glycosidase significantly reduced infection success, and implicated algal cell surface glycans in recognition. Using flow cytometry, alpha-mannose/alpha-glucose and alpha-galactose residues were identified as potential recognition ligands on the algal cell surface. Finally, inhibition of these cell surface glycans significantly reduced infection of F. scutaria larvae by the algae. These data provide evidence that the algal cell surface contains glycan ligands, such as alpha-mannose/alpha-glucose and alpha-galactose, which play a role in recognition during initial contact at the onset of symbiosis with F. scutaria larvae.

Oral ingestion of mannose alters the expression level of deaminoneuraminic acid (KDN) in mouse organs.

Deaminoneuraminic acid (KDN) is a unique member of the sialic acid family. We previously demonstrated that free KDN is synthesized de novo from mannose as its precursor sugar in trout testis, and that the amount of intracellular KDN increases in mouse B16 melanoma cells cultured in mannose-rich media [Angata et al. (1999) J. Biol. Chem. 274, 22949-56; Angata et al. (1999) Biochem. Biophys. Res. Commun. 261, 326-31]. In the present study, we first demonstrated a mannose-induced increase in intracellular KDN in various cultured mouse and human cell lines. These results led us to examine whether KDN expression in mouse organs is altered by exogenously administered mannose. Under normal feeding conditions, intracellular free KDN was present at very low levels (19-48 pmol/mg protein) in liver, spleen, and lung, and was not detected in kidney or brain. Oral ingestion of mannose, both short-term (90 min) and long-term (2 wk), resulted in an increase of intracellular KDN up to 60-81 pmol/mg protein in spleen and lung and 6.9-18 pmol/mg protein in kidney and brain; however, no change was observed in liver. The level of KDN in organs appears not to be determined only by the KDN 9-phosphate synthase activity, but might also be affected by other enzymes that utilize mannose 6-phosphate as a substrate as well as the enzymes that breakdown KDN, like KDN-pyruvate lyase. In blood, the detectable amount of free KDN did not change on oral ingestion of mannose. These findings indicate that mannose in the diet affects KDN metabolism in various organs, and provide clues to the mechanism of altered KDN expression in some tumor cells and aged organs.

Influence of the endosymbiont of Blastocrithidia culicis and Crithidia deanei on the glycoconjugate expression and on Aedes aegypti interaction.

Blastocrithidia culicis and Crithidia deanei are trypanosomatid protozoa of insects that normally contain intracellular symbiotic bacteria. The protozoa can be rid of their endosymbionts by antibiotics, producing a cured cell line. Here, we analyzed the glycoconjugate profiles of endosymbiont-harboring and cured strains of B. culicis and C. deanei by Western blotting and flow cytometry analyses using lectins that recognize specifically sialic acid and mannose-like residues. The absence of the endosymbiont increased the intensity of the lectins binding on both trypanosomatids. In addition, wild and cured strain-specific glycoconjugate bands were identified. The role of the surface saccharide residues on the interaction with explanted guts from Aedes aegypti gut was assessed. The aposymbiotic strains of B. culicis and C. deanei presented interaction rates 3.3- and 2.3-fold lower with the insect gut, respectively, when compared with the endosymbiont-bearing strains. The interaction rate of sialidase-treated cells of the wild and cured strains of B. culicis and C. deanei was reduced in at least 90% in relation to the control. The interaction of B. culicis (wild strain) with explanted guts was inhibited in the presence of mucin (56%), fetuin (62%), sialyllactose (64%) and alpha-methyl-D-mannoside (80%), while in C. deanei (wild strain) the inhibition was 53%, 56%, 79% and 34%, respectively. Collectively, our results suggest a possible involvement of sialomolecules and mannose-rich glycoconjugates in the interaction between insect trypanosomatids and the invertebrate host.

Activation of cellular functions in macrophages by venom secretory Asp-49 and Lys-49 phospholipases A(2).

The in vitro effects of myotoxin III (MT-III), an Asp-49 catalytically-active phospholipase A(2), and myotoxin II (MT-II), a catalytically-inactive Lys-49 variant, isolated from Bothrops asper snake venom, on phagocytosis and production of hydrogen peroxide (H(2)O(2)) by thioglycollate-elicited macrophages were investigated. MT-II and MT-III were cytotoxic to mouse peritoneal macrophages at concentrations higher than 25 microg/ml. At non-cytotoxic concentrations, MT-II stimulated Fcgamma, complement, mannose and beta-glucan receptors-mediated phagocytosis, whereas MT-III stimulated only the mannose and beta-glucan receptors-mediated phagocytosis. Moreover, both myotoxins induced the release of H(2)O(2) by thioglycollate-elicited macrophages, MT-III being the most potent stimulator. MT-II induced the release of H(2)O(2) only at a concentration of 3.2 microg/ml (130% increment) while MT-III induced this effect at all concentrations tested (0.5-2.5 microg/ml; average of 206% increment). It is concluded that, at non-cytotoxic concentrations, MT-II and MT-III activate defense mechanisms in macrophages up regulating phagocytosis, mainly via mannose and beta-glucan receptors, and the respiratory burst.

Evaluación de la reacción acrosomal en espermatozoides humanos inducida por los monosacáridos manosa y N-acetilglucosamina / Evaluation of the role of the monosaccharides, mannose and N-acetylglucosamine in the induction of the acrosome reaction in human spermatozoa

Objetivo: El objetivo de esta investigación fue evaluar el papel de los monosacáridos, manosa y Nacetilglucosamina en la inducción de la reacción acrosomal en espermatozoides humanos. Método: Cada individuo donó una muestra de semen, la cual fue sometida al proceso de capacitación usando albúmina sérica bovina, posterior a lo cual se evaluó el porcentaje de espermatozoides que presentaban reacción acrosomal al estimularlos con manosa y N-acetilglucosamina, usando microscopía de fluorescencia y citometría de flujo. Resultados: Los resultados obtenidos en las 10 muestras evaluadas mediante citometría de flujo mostraron que sólo el ionóforo de calcio A23187 indujo reacción acrosomal (p<0,001); en contraste por microscopía de fluorescencia usando la lectina PSA-FITC, se observó un aumento estadísticamente significativo en la reacción acrosomal inducida con manosa y N-acetilglucosamina (p<0,001). Conclusiones: Los monosacáridos manosa y N-acetilglucosamina inducen reacción acrosomal parcial y no completa, por lo tanto el espermatozoide gracias a la reacción acrosomal parcial y a las modificaciones morfológicas que sufre durante ese proceso es capaz de fertilizar el oocito (AU)

Objective: The aim of this investigation was to evaluate the role of the monosaccharides, mannose and N-acetylglucosamine in the induction of the acrosome reaction in human spermatozoa. Methods: Each individual gave a semen sample, the samples were capacitated using bovien serum albumin the analysis of the mannose and N-acetylglucosamine induced acrosome reaction was evaluated for fluorescence microscopy and flow cytometry. Results: The results obtained in the acrosomal reaction in the 10 samples evaluated by flow cytometry showed that only calcium ionophore A23187 induced acrosomal reaction (p<0.001); in contrast, by fluorescence microscopy, was observed a statistically significant increase in the induced acrosomal reaction with mannose and N-acetylglucosamine (p<0.001). Conclusions: The monosaccharides mannose and N-acetylglucosamine induces partial acrosomal reaction and it does not complete, therefore the spermatozoa due to partial acrosomal reaction and the morphologics modifications that suffer during that process is able to fertilize the oocyte (AU)

Mannose-containing molecular patterns are strong inducers of cyclooxygenase-2 expression and prostaglandin E2 production in human macrophages.

The induction of cyclooxygenase-2 (COX-2) and the production of PGE(2) in response to pathogen-associated molecular patterns decorated with mannose moieties were studied in human monocytes and monocyte-derived macrophages (MDM). Saccharomyces cerevisiae mannan was a robust agonist, suggesting the involvement of the mannose receptor (MR). MR expression increased along the macrophage differentiation route, as judged from both its surface display assessed by flow cytometry and the ability of MDM to ingest mannosylated BSA. Treatment with mannose-BSA, a weak agonist of the MR containing a lower ratio of attached sugar compared with pure polysaccharides, before the addition of mannan inhibited COX-2 expression, whereas this was not observed when agonists other than mannan and zymosan were used. HeLa cells, which were found to express MR mRNA, showed a significant induction of COX-2 expression upon mannan challenge. Conversely, mannan did not induce COX-2 expression in HEK293 cells, which express the mRNA encoding Endo180, a parent receptor pertaining to the MR family, but not the MR itself. These data indicate that mannan is a strong inducer of COX-2 expression in human MDM, most likely by acting through the MR route. Because COX-2 products can be both proinflammatory and immunomodulatory, these results disclose a signaling route triggered by mannose-decorated pathogen-associated molecular patterns, which can be involved in both the response to pathogens and the maintenance of homeostasis.

Role of glycosylation in development.

Researchers have long predicted that complex carbohydrates on cell surfaces would play important roles in developmental processes because of the observation that specific carbohydrate structures appear in specific spatial and temporal patterns throughout development. The astounding number and complexity of carbohydrate structures on cell surfaces added support to the concept that glycoconjugates would function in cellular communication during development. Although the structural complexity inherent in glycoconjugates has slowed advances in our understanding of their functions, the complete sequencing of the genomes of organisms classically used in developmental studies (e.g., mice, Drosophila melanogaster, and Caenorhabditis elegans) has led to demonstration of essential functions for a number of glycoconjugates in developmental processes. Here we present a review of recent studies analyzing function of a variety of glycoconjugates (O-fucose, O-mannose, N-glycans, mucin-type O-glycans, proteoglycans, glycosphingolipids), focusing on lessons learned from human disease and genetic studies in mice, D. melanogaster, and C. elegans.

N-linked sialyated sugar receptors support haematopoietic cell-osteoblast adhesions.

Haematopoietic progenitor cells proliferate and develop predominantly when they adhere to bone marrow stromal cells such as osteoblasts. Therefore, changes in adhesion may be a common mechanism by which stem cells survive, mature and properly traffic between the bone marrow and the circulation. To characterize these adhesion molecules, we reduced the bone marrow cavity to a simple adhesion assay between KG1a (a CD34+ haematopoietic cell line) and osteosarcoma monolayers (MG-63 or SaOS-2). The data demonstrated that adhesion was mediated by cell-to-cell rather than cell-to-matrix contact, was sensitive to trypsin, calcium chelators and glycosylation inhibitors. Selective pretreatment attributed the constitutive binding to N-linked glycans on KG1a. When carboprocessing was inhibited later at the high mannose intermediate (via deoxymannojirimycin), adhesion was retained. Surprisingly, binding of KG1a to SaOS-2 increased past constitutive levels as doses of tunicamycin or deoxymannojirimycin dropped. Selective pretreatment suggested that this 'inducible' binding resides with molecule(s) on SaOS-2. If the terminal sialic acid was digested (via neuraminidase), this induced response was duplicated. These data, verified in primary cells, suggest that the initial tethering between blood and bone cells in this model is probably due to heavily glycosylated, rapidly processed protein(s) on both cell types.

High mannose glycans and sialic acid on gp120 regulate binding of mannose-binding lectin (MBL) to HIV type 1.

Mannose-binding lectin (MBL) is a C-type lectin of the innate immune system that binds to carbohydrates on the surface of certain microorganisms. Previous studies showed that MBL binds to gp120, the envelope glycoprotein of HIV-1. gp120 is extensively glycosylated, with N-linked complex and high mannose carbohydrates accounting for about half of the molecular weight. The objectives of this study were to determine the types of glycans on gp120 important for MBL binding and to determine if alteration of complex glycans with neuraminidase (NA) could enhance the interaction of MBL with virus. Lectin blot analyses revealed that MBL interacted with recombinant gp120 (rgp120) from both T cell-tropic and M-tropic virus strains. Treatment of rgp120 with endoglycosidase H (eH) or endoglycosidase F1 (eF1) abrogated binding of MBL, but did not decrease binding of wheat germ agglutinin indicating that high mannose and/or hybrid N-linked glycans were required for MBL binding. Removal of sialic acids from rgp120 with NA enhanced MBL binding. Treatment of intact virus from T cell lines or primary isolates with eF1 also significantly decreased HIV binding to MBL, while treatment with NA substantially increased binding. Treatment of virus with both eF1 and NA did not decrease binding compared to NA alone suggesting that NA treatment exposed binding sites on gp120 that are not high mannose glycans. These studies provide evidence that MBL binds to HIV via high mannose carbohydrates on gp120 and shows that the interaction of MBL with virus is regulated by sialylation.

Chaperone-like functions of high-mannose type and complex-type N-glycans and their molecular basis.

It has recently become apparent that high-mannose type N-glycans directly promote protein folding, whereas complex-type ones play a crucial role in the stabilization of protein functional conformations through hydrophobic interactions with the hydrophobic protein surfaces. Here an attempt was made to understand more deeply the molecular basis of these chaperone-like functions with the aid of information obtained from spacefill models of N-glycans. The promotion of protein folding by high-mannose N-glycans seemed to be based on their unique structure, which includes a hydrophobic region similar to the cyclodextrin cavity. The promotive features of high-mannose N-glycans newly observed under various conditions furnished strong support for the view that both intra- and extramolecular high-mannose N-glycans are directly involved in the promotion of protein folding in the endoplasmic reticulum. Further, it was revealed that the N-acetyllactosamine units in complex-type N-glycans have an amphiphilic structure and greatly contribute to the formation of extensive hydrophobic surfaces and, consequently, to the N-glycan-protein hydrophobic interactions. The processing of high-mannose type N-glycans to complex-type ones seems to be an ingenious device to enable the N-glycans to perform these two chaperone-like functions.

Secretion of phosphomannosyl-deficient arylsulphatase A and cathepsin D from isolated human macrophages.

The transfer of macrophage-secreted arylsulphatase A (ASA) to enzyme-deficient brain cells is part of the therapeutic concept of bone marrow transplantation in lysosomal storage diseases. Here we have investigated this transfer in vitro. The uptake of (125)I-labelled recombinant human ASA purified from ASA-overexpressing mouse embryonic fibroblasts deficient for mannose 6-phosphate (M6P) receptors in a mouse ASA-deficient astroglial cell line was completely inhibited by M6P. In contrast, when ASA-deficient astroglial cells were incubated with secretions of [(35)S]methionine-labelled human macrophages or mouse microglia, containing various lysosomal enzymes, neither ASA nor cathepsin D (CTSD) were detected in acceptor cells. Co-culturing of metabolically labelled macrophages with ASA-deficient glial cells did not result in an M6P-dependent transfer of ASA or CTSD between these two cell types. In secretions of [(33)P]phosphate-labelled macrophages no or weakly phosphorylated ASA and CTSD precursor polypeptides were found, whereas both intracellular and secreted ASA from ASA-overexpressing baby hamster kidney cells displayed (33)P-labelled M6P residues. Finally, the uptake of CTSD from secretions of [(35)S]methionine-labelled macrophages in rat hepatocytes was M6P-independent. These data indicated that lysosomal enzymes secreted by human macrophages or a mouse microglial cell line cannot be endocytosed by brain cells due to the failure to equip newly synthesized lysosomal enzymes with the M6P recognition marker efficiently. The data suggest that other mechanisms than the proposed M6P-dependent secretion/recapture of lysosomal enzymes might be responsible for therapeutic effects of bone marrow transplantation in the brain.