scholarly journals Phagocytosis stimulates alternative glycosylation of macrosialin (mouse CD68), a macrophage-specific endosomal protein

1999 ◽  
Vol 338 (3) ◽  
pp. 687-694 ◽  
Author(s):  
Rosângela P. da SILVA ◽  
Siamon GORDON
Keyword(s):  
Protein Synthesis ◽  
Sialic Acid ◽  
Membrane Protein ◽  
Lectin Binding ◽  
Peritoneal Macrophages ◽  
Binding Activity ◽  
Amaranthus Caudatus ◽  
Galanthus Nivalis ◽  
Particle Internalization

Macrosialin (mouse CD68), a macrophage-specific member of the lysosomal-associated membrane protein family, displays N-linked glycosylation and a heavily sialylated, mucin-like domain. We show that phagocytosis of zymosan by inflammatory peritoneal macrophages potently alters glycan processing of macrosialin in vitro. The phagocytic glycoform is not induced by other forms of endocytosis and depends on particle internalization. Zymosan uptake does not influence macrosialin protein synthesis, but increases the specific incorporation of d-[2-3H]mannose, d-[6-3H]galactose, N-acetyl-d-[1-3H]glucosamine and l-[5,6-3H]fucose by 2–15-fold. The phagocytic glycoform displays increased binding of agglutinins from peanut, Amaranthus caudatus and Galanthus nivalis, whereas binding of the sialic-acid-specific Maakia amurensis agglutinin is slightly reduced. Digestion by N-Glycanase abolishes the incorporation of [3H]mannose label and Galanthus nivalis agglutinin binding activity, but preserves the incorporation of galactose and N-acetylglucosamine and specific lectin binding. We also show that phagocytosis increases the complexity and length of O-linked chains. The data presented highlight the importance of differential glycosylation in the biology of macrosialin, phagosomes and macrophages in general.

scholarly journals In vitro membrane protein synthesis inside Sec translocon-reconstituted cell-sized liposomes

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Naoki Ohta ◽  
Yasuhiko Kato ◽  
Hajime Watanabe ◽  
Hirotada Mori ◽  
Tomoaki Matsuura
Keyword(s):  
Protein Synthesis ◽  
Membrane Protein ◽  
Sec Translocon

Terminal α2-6 Linked Sialic Acid Expression On VWF Specifically Enhances Proteolysis by ADAMTS13.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 30-30
Author(s):  
Rachel Therese McGrath ◽  
Thomas A J McKinnon ◽  
Barry Byrne ◽  
Richard O'Kennedy ◽  
Michael Laffan ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Blood Group ◽  
Conflicts Of Interest ◽  
Lectin Binding ◽  
Gel Filtration ◽  
Specific Effect ◽  
Cysteine Proteases ◽  
Binding Activity ◽  
Collagen Binding ◽  
Sodium Metaperiodate

Abstract Abstract 30 VWF is a large plasma sialoglycoprotein that mediates platelet tethering at sites of vascular injury. VWF function is dependent upon VWF multimeric composition, which is regulated in plasma by ADAMTS13. ABO(H) blood group determinants expressed on VWF N-linked glycans significantly influence susceptibility to ADAMTS13 proteolysis. In this study, we investigated whether terminal sialic acid residues expressed on the N- and O-linked glycans of VWF may also regulate proteolysis by ADAMTS13. VWF was initially purified from human plasma (pdVWF) by cryoprecipitation and gel filtration. Subsequently, VWF sialylation was modified using specific exoglycosidases and quantified by lectin-binding ELISA. The rate of glycosidase-treated VWF proteolysis by ADAMTS13 was determined by incubation with recombinant ADAMTS13 and subsequent measurement of residual VWF collagen binding activity. Complete VWF deglycosylation has been shown to enhance the rate of proteolysis by ADAMTS13. In contrast, enzymatic desialylation of VWF by α2-3,6,8,9 neuraminidase (Neu-VWF) markedly impaired the rate of ADAMTS13-mediated VWF proteolysis. Neu-VWF collagen binding activity was reduced to only 50±14% by ADAMTS13, compared to 11±7% for untreated VWF (p<0.01) at the same time point. Despite this, Neu-VWF exhibited increased susceptibility to proteases other than ADAMTS13 (trypsin, chymotrypsin and cathepsin B; all p<0.05). VWF sialylation is therefore a specific enhancer of ADAMTS13-mediated proteolysis. Consequently, quantification and molecular distribution of VWF sialylation was examined by sequential digestion and HPLC analysis. Total sialic acid expression on pdVWF was 167nmol/mg, of which the majority (133.4nmol/mg or 80.1%) was present on its N-linked glycan chains. Interestingly, despite the resistance to ADAMTS13 proteolysis observed upon complete desialylation, digestion of pdVWF with α2-3 neuraminidase to remove predominantly O-linked sialic acid did not influence the rate of ADAMTS13 proteolysis. Previous studies have demonstrated that VWF expressing different blood groups exhibit altered rates of proteolysis by ADAMTS13 (O ≥ B > A ≥ AB). Since α2-6 linked sialic acid and ABO(H) determinants are both expressed as terminal antigens on VWF N-linked glycans, the effect of desialylation upon blood group-specific VWF proteolysis by ADAMTS13 was determined. As expected, untreated group O VWF was cleaved significantly faster than group AB-VWF (p<0.05). However, the ability of ABO blood group to regulate ADAMTS13 proteolysis was completely ablated upon VWF desialylation, as both Neu-O-VWF and Neu-AB-VWF were cleaved by ADAMTS13 at identical rates. This indicates that VWF sialylation constitutes a more important determinant of susceptibility to ADAMTS13 proteolysis than ABO(H) expression. Sialic acid can mediate protein-protein interactions through either conformational and/or charge-mediated mechanisms. Despite this, sodium metaperiodate treatment of pdVWF to remove sialic acid anionic charge did not influence the rate of proteolysis by ADAMTS13. In contrast, the ability of sialic acid to specifically enhance ADAMTS13 proteolysis of VWF was significantly attenuated at high urea concentrations (≥2M), supporting the hypothesis that VWF sialylation enhances proteolysis by ADAMTS13 by promoting a ADAMTS13-specific permissive conformation. These novel data demonstrate that although sialic acid protects VWF against proteolysis by serine and cysteine proteases, it also specifically enhances susceptibility to proteolysis by ADAMTS13. Moreover, the magnitude of this sialic acid-specific effect on VWF proteolysis by ADAMTS13 is more marked than that attributable to N-linked ABO(H) blood group antigen expression. Therefore, quantitative variation in VWF sialylation represents a key regulator of VWF multimeric composition, and as such, is likely to be of clear patho-physiological significance. Disclosures: No relevant conflicts of interest to declare.

Direct preparation of giant proteo-liposomes by in vitro membrane protein synthesis

2008 ◽  
Vol 133 (2) ◽  
pp. 190-195 ◽  
Author(s):  
Shin-ichiro M. Nomura ◽  
Satoshi Kondoh ◽  
Wakiko Asayama ◽  
Akikazu Asada ◽  
Shigemichi. Nishikawa ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Membrane Protein ◽  
Direct Preparation

scholarly journals A membrane-bound form of glutamate dehydrogenase possesses an ATP-dependent high-affinity microtubule-binding activity

1993 ◽  
Vol 295 (2) ◽  
pp. 447-455 ◽  
Author(s):  
F Rajas ◽  
B Rousset
Keyword(s):  
Membrane Protein ◽  
Glutamate Dehydrogenase ◽  
Rat Liver ◽  
Binding Activity ◽  
Binding Studies ◽  
Microtubule Binding ◽  
Phase Partitioning ◽  
Membrane Bound ◽  
Commercial Sources

We previously identified a 50 kDa membrane protein which bound to in vitro assembled microtubules [Mithieux and Rousset (1989) J. Biol. Chem. 264, 4664-4668]. This protein exhibited the expected properties for mediating the ATP-dependent association of vesicles with microtubules [Mithieux, Audebet and Rousset (1988) Biochim. Biophys. Acta 969, 121-130]. The 50 kDa membrane protein (MP50), initially extracted in very low amount from isolated pig thyroid lysosomes/endosomes, has now been purified from membrane preparations of crude vesicle fractions from pig liver and brain. MP50 was isolated from detergent-solubilized membrane protein by affinity chromatography on immobilized ATP; 3-5 mg of MP50 was obtained from 100 g of liver tissue. Phase partitioning in Triton X-114 indicated that MP50 is a peripheral membrane protein. Radioiodinated liver MP50 bound to microtubules assembled in vitro. The binding was inhibited by ATP (Ki = 0.76 mM) and displaced by unlabelled liver or brain MP50. Equilibrium binding studies yielded KD values of 1.8 x 10(-7) M. By N-terminal amino acid sequence analysis, MP50 was identified as glutamate dehydrogenase (GDH), by comparison of V8 protease peptide maps of MP50 with purified liver GDH. Liver MP50 exhibited a low GDH activity; 4-5 units/mg compared with 18 and 34 units/mg for purified bovine and rat liver GDH respectively. Bovine and rat liver GDH yielded six spots from pI 5.7 to 7.2 when analysed by two-dimensional electrophoresis; in contrast, MP50 gave one main spot (corresponding to spot 2 of liver GDH) with a pI of approx. 6.5. Soluble liver GDH from commercial sources exhibited a very low or no microtubule-binding activity. In conclusion, we have found a membrane-bound form of GDH capable of specific and nucleotide-sensitive interaction with microtubules. Our data suggest that GDH isoproteins, the number of which has been undervalued up to now, could have cellular functions other than that of an enzyme.

In Vitro Membrane Protein Synthesis Inside Cell-Sized Vesicles Reveals the Dependence of Membrane Protein Integration on Vesicle Volume

2013 ◽  
Vol 3 (6) ◽  
pp. 372-379 ◽  
Author(s):  
Haruka Soga ◽  
Satoshi Fujii ◽  
Tetsuya Yomo ◽  
Yasuhiko Kato ◽  
Hajime Watanabe ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Membrane Protein

scholarly journals Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression

Biochemistry ◽  
2016 ◽  
Vol 55 (30) ◽  
pp. 4212-4219 ◽  
Author(s):  
Paul J. Focke ◽  
Christopher Hein ◽  
Beate Hoffmann ◽  
Kimberly Matulef ◽  
Frank Bernhard ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Membrane Protein ◽  
Protein Expression ◽  
Free Protein ◽  
Membrane Protein Expression ◽  
Cell Free Protein Synthesis

scholarly journals An L-arginine-dependent mechanism mediates kupffer cell inhibition of hepatocyte protein synthesis in vitro

1989 ◽  
Vol 169 (4) ◽  
pp. 1467-1472 ◽  
Author(s):  
TR Billiar ◽  
RD Curran ◽  
DJ Stuehr ◽  
MA West ◽  
BG Bentz ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Peritoneal Macrophages ◽  
Metabolic Responses ◽  
Leucine Incorporation ◽  
Target Cells ◽  
Microbial Invasion ◽  
Nitrite Nitrate ◽  
Dependent Mechanism

The hepatic failure associated with severe sepsis is characterized by specific, progressive, and often irreversible defects in hepatocellular metabolism (1). Although the etiologic microbe can often be identified, the direct causes and mechanisms of the hepatocellular dysfunction are poorly understood. We have hypothesized that Kupffer cells (KC), which interact with ambient septic stimuli, respond by providing signals to adjacent hepatocytes (HC) in sepsis . Furthermore, we have provided evidence (2, 3) that KC activated by LPS from Gram-negative bacteria can induce profound changes in the function of neighboring HC in coculture. In our model, coculture of either KC (2) or peritoneal macrophages (Mφ)(3) with HC normally promotes HC protein synthesis ([(3)H]leucine incorporation). The addition of LPS or killed Escherichia colt' to such cocultures induces a profound decrease in HC protein synthesis, as well as qualitative changes ([(35)S]methionine, SDS-gel electrophoresis) in protein synthesis without inducing HC death (2, 3) . In this report we show that the inhibition in protein synthesis is mediated via an L-arginine-dependent mechanism. The metabolism of L-arginine by activated Mφ to substances with cytostatic and even lethal effects on target cells is a relatively recent discovery. After the description by Stuehr and Marletta (4, 5) that LPS- triggered Mφ produced nitrite/nitrate (NO(2)(-)/NO(3)(-)), Hibbs et al. (6, 7) and Iyengar et al. (8) demonstrated that L-arginine was the substrate for the formation of both these nitrogen end products and citrulline. A role for the arginine-dependent mechanism in Mφ tumor cytotoxicity (6, 7) and microbiostatic activity (9) has been suggested. However, the in vivo functions of this novel Mφ mechanism have not yet been defined, but it is possible that there are both physiologic as well as pathologic roles. Our in vitro results raise the possibility that some metabolic responses to microbial invasion maybe partially mediated by the L-arginine-dependent mechanism. What other metabolic responses are affected and the possible pathologic consequences remain to be studied.

Lectin binding on surfaces of Frankia strains

1983 ◽  
Vol 61 (11) ◽  
pp. 2889-2897 ◽  
Author(s):  
Annie Chaboud ◽  
Maurice Lalonde
Keyword(s):  
Sialic Acid ◽  
Lectin Binding ◽  
Carbohydrate Binding ◽  
Symbiotic Association ◽  
Cell Surfaces ◽  
Species Definition ◽  
Sialic Acid Binding ◽  
Host Plant Specificity ◽  
Binding Lectin

As a prerequisite for the formation of symbiotic association the two partners must come in contact at their cell surfaces, where the phenomena of specificity and recognition are believed to take place. Therefore, in the case of actinorhizal symbioses, probing of Frankia cell surfaces was investigated with fluorescein-labelled lectins as specific markers for sugar residues. Eleven Frankia isolates grown in vitro and originating from Alnus and Elaeagnus host-plant specificity groups were tested with lectins of different carbohydrate-binding specificities. The N-acetylglucosamine and sialic acid binding lectin groups were able to bind to cell surfaces of both type-N and type-P strains of the Alnus group but not to any Frankia strains of the Elaeagnus group. Sugar residues recognized by these lectin groups on colonies of Frankia strains of the Alnus group were detected on hyphae, vesicles, and sporangia but not on mature spores. The present lectin binding study demonstrates that the use of lectins as bioprobes can be a valid tool in the awaited species definition in the genus Frankia.

scholarly journals Siglec-E retards atherosclerosis by inhibiting CD36-mediated foam cell formation

2021 ◽  
Vol 28 (1) ◽  
Author(s):  
Yaw-Wen Hsu ◽  
Fu-Fei Hsu ◽  
Ming-Tsai Chiang ◽  
Dong-Lin Tsai ◽  
Fu-An Li ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Cell Surface ◽  
Proteomic Analysis ◽  
Foam Cell ◽  
Oxidized Ldl ◽  
Cell Formation ◽  
Peritoneal Macrophages ◽  
Foam Cell Formation ◽  
Ldl Uptake

Abstract Background The accumulation of lipid-laden macrophages, foam cells, within sub-endothelial intima is a key feature of early atherosclerosis. Siglec-E, a mouse orthologue of human Siglec-9, is a sialic acid binding lectin predominantly expressed on the surface of myeloid cells to transduce inhibitory signal via recruitment of SH2-domain containing protein tyrosine phosphatase SHP-1/2 upon binding to its sialoglycan ligands. Whether Siglec-E expression on macrophages impacts foam cell formation and atherosclerosis remains to be established. Methods ApoE-deficient (apoE−/−) and apoE/Siglec-E-double deficient (apoE−/−/Siglec-E−/−) mice were placed on high fat diet for 3 months and their lipid profiles and severities of atherosclerosis were assessed. Modified low-density lipoprotein (LDL) uptake and foam cell formation in wild type (WT) and Siglec-E−/−- peritoneal macrophages were examined in vitro. Potential Siglec-E-interacting proteins were identified by proximity labeling in conjunction with proteomic analysis and confirmed by coimmunoprecipitation experiment. Impacts of Siglec-E expression and cell surface sialic acid status on oxidized LDL uptake and signaling involved were examined by biochemical assays. Results Here we show that genetic deletion of Siglec-E accelerated atherosclerosis without affecting lipid profile in apoE−/− mice. Siglec-E deficiency promotes foam cell formation by enhancing acetylated and oxidized LDL uptake without affecting cholesterol efflux in macrophages in vitro. By performing proximity labeling and proteomic analysis, we identified scavenger receptor CD36 as a cell surface protein interacting with Siglec-E. Further experiments performed in HEK293T cells transiently overexpressing Siglec-E and CD36 and peritoneal macrophages demonstrated that depletion of cell surface sialic acids by treatment with sialyltransferase inhibitor or sialidase did not affect interaction between Siglec-E and CD36 but retarded Siglec-E-mediated inhibition on oxidized LDL uptake. Subsequent experiments revealed that oxidized LDL induced transient Siglec-E tyrosine phosphorylation and recruitment of SHP-1 phosphatase in macrophages. VAV, a downstream effector implicated in CD36-mediated oxidized LDL uptake, was shown to interact with SHP-1 following oxidized LDL treatment. Moreover, oxidized LDL-induced VAV phosphorylation was substantially lower in WT macrophages comparing to Siglec-E−/− counterparts. Conclusions These data support the protective role of Siglec-E in atherosclerosis. Mechanistically, Siglec-E interacts with CD36 to suppress downstream VAV signaling involved in modified LDL uptake.

Loss Of Platelet Membrane Glycoproteins Or Terminal Sialic Acid Detected By Fitc-Lectins

1981 ◽  
Author(s):  
L McGregor ◽  
J L McGregor ◽  
K J Clemetson ◽  
M Dechavanne ◽  
E F Lüscher
Keyword(s):  
Sialic Acid ◽  
Ricinus Communis ◽  
Lectin Binding ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Binding Studies ◽  
Platelet Surface ◽  
Terminal Sialic Acid

Pre-thrombic conditions in certain individuals resulting from enhanced platelet-vessel wall or platelet-platelet interactions are perhaps characterized by a reduction in certain membrane glycoproteins or loss of terminal sialic acid. In order to investigate if such changes are detectable, the binding of FITC-lectins to human platelets treated under in vitro conditions with certain proteases to mimic possible in vivo changes occuring on the platelet surface, has been examined. Human platelets were isolated, washed and either treated with neuraminidase (10 U) or plasmin (1 CU) before fixing with formaldehyde. Binding studies were performed by the method of Monsigny et al. using FITC labelled wheat germ agglutinin (WGA), Lens culinaris lectin (LCL), Ricinus communis agglutinin (RCA) and concanavalin A (ConA). The number of lectin-binding sites (n) and the dissociation constant (Kd) were obtained by Steck and Wallach reciprocal plots. After neuraminidase or plasmin treatment n was reduced but Kd remained approximately the same with WGA. FITC-RCA-60 gave a slight fluorescence with untreated and very strong fluorescence with neuraminidase treated platelets. Platelet glycoproteins separated by 2-dimensional gel electrophoresis were identified by binding of fluorescent lectins. Plasmin decreased the intensity of GP Ib and IIb and removed Ia completely. Neuraminidase decreased the labelling of Ib by WGA. These techniques show promise as methods of detecting pre-thrombotic conditions.

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