The effects of digestive enzymes on characteristics of placental insulin receptor. Comparison of particulate and soluble receptor preparations.
The role of the surrounding membrane structure on the binding characteristics of the insulin receptor was studied by using several digestive enzymes. The effects observed with particulate membrane preparations are compared with those from soluble receptor preparations. beta-Galactosidase and neuraminidase had no effect on insulin binding to either particulate or soluble receptors from human placentae. Exposure to 2 units of phospholipase C/ml increased insulin binding to particulate membranes, but was without effect on the soluble receptor preparation. The increase in binding to particulate membranes was shown to be due to an increase in apparent receptor number. After 5 min exposure to 500 microgram of trypsin/ml there was an increase in insulin binding to the particulate membrane fraction, owing to an increase in receptor affinity. After 15 min exposure to this amount of trypsin, binding decreased, owing to a progressive decrease in receptor availability. In contrast, this concentration of trypsin had no effect on the solubilized receptor preparation. Because of the differential effects of phospholipase C and trypsin on the particulate compared with the solubilized receptor preparations, it is concluded that the effects of these enzymes were due to an effect on the surrounding membrane structure. Changes in receptor configuration due to alterations within the adjoining membrane provide a potential mechanism for mediating short-term alterations in receptor function. (+info)
Cell surface sialic acid and the regulation of immune cell interactions: the neuraminidase effect reconsidered.
It has been known for over a decade that sialidase (neuraminidase) treatment could substantially enhance the capacity of resting B cells to stimulate the proliferation of allogeneic and antigen specific, syngeneic T cells. Thus, cell-surface sialic acid was implicated as a potential modulator of immune cell interaction. However, little progress has been made in either identifying explicit roles for sialic acid in this system or in hypothesizing mechanisms to explain the "neuraminidase effect." Here we show for the first time that cell surface sialic acid on medium incubated B cells blocks access to costimulatory molecules on the B cell surface, and that this is the most likely explanation for the neuraminidase effect. Further, we show that it is likely to be upregulation of ICAM-1 and its subsequent engagement of LFA-1 rather than loss of cell surface sialic acid that in part regulates access to CD86 and other costimulatory molecules. However, we cannot exclude a role for CD86-bound sialic acid on the B cell in modulating binding to T cell CD28. Because sialidase treatment of resting B cells but not resting T cells enables T cell activation, we suggest that sialidase treatment may still be an analogue for an authentic step in B cell activation, and show that for highly activated B cells (activated with polyclonal anti-IgM plus INF-gamma) there is specific loss 2, 6-linked sialic acid. Potential roles for sialic acid in modulating B cell/T cell collaboration are discussed. (+info)
N-Linked glycosylation and sialylation of the acid-labile subunit. Role in complex formation with insulin-like growth factor (IGF)-binding protein-3 and the IGFs.
Over 75% of the circulating insulin-like growth factors (IGF-I and -II) are bound in 140-kDa ternary complexes with IGF-binding protein-3 (IGFBP-3) and the 84-86-kDa acid-labile subunit (ALS), a glycoprotein containing 20 kDa of carbohydrate. The ternary complexes regulate IGF availability to the tissues. Since interactions of glycoproteins can be influenced by their glycan moieties, this study aimed to determine the role of ALS glycosylation in ternary complex formation. Complete deglycosylation abolished the ability of ALS to associate with IGFBP-3. To examine this further, seven recombinant ALS mutants each lacking one of the seven glycan attachment sites were expressed in CHO cells. All the mutants bound IGFBP-3, demonstrating that this interaction is not dependent on any single glycan chain. Enzymatic desialylation of ALS caused a shift in isoelectric point from 4.5 toward 7, demonstrating a substantial contribution of anionic charge by sialic acid. Ionic interactions are known to be involved in the association between ALS and IGFBP-3. Desialylation reduced the affinity of ALS for IGFBP-3. IGF complexes by 50-80%. Since serum protein glycosylation is often modified in disease states, the dependence of IGF ternary complex formation on the glycosylation state of ALS suggests a novel mechanism for regulation of IGF bioavailability. (+info)
Binding partners for the myelin-associated glycoprotein of N2A neuroblastoma cells.
The myelin-associated glycoprotein (MAG) has been proposed to be important for the integrity of myelinated axons. For a better understanding of the interactions involved in the binding of MAG to neuronal axons, we performed this study to identify the binding partners for MAG on neuronal cells. Experiments with glycosylation inhibitors revealed that sialylated N-glycans of glycoproteins represent the major binding sites for MAG on the neuroblastoma cell line N2A. From extracts of [3H]glucosamine-labelled N2A cells several glycoproteins with molecular weights between 20 and 230 kDa were affinity-precipitated using immobilised MAG. The interactions of these proteins with MAG were sialic acid-dependent and specific for MAG. (+info)
Carbohydrate on human factor VIII/von Willebrand factor. Impairment of function by removal of specific galactose residues.
Human factor VIII/von Willebrand factor protein containing 120 +/- 12 nmol of sialic acid and 135 +/- 13 nmol of galactose/mg of protein was digested with neuraminidase. The affinity of native factor VIII/von Willebrand factor and its asialo form for the hepatic lectin that specifically binds asialoglycoproteins was assessed from in vitro binding experiments. Native factor VIII/von Willebrand factor exhibited negligible affinity while binding of the asialo derivative was comparable to that observed for asialo-alpha1-acid glycoprotein. Incubation of asialo-factor VIII/von Willebrand factor with Streptococcus pneumoniae beta-galactosidase removed only 62% of the galactose but abolished binding to the purified hepatic lectin. When the asialo derivative was incubated with purified beta-D-galactoside alpha2 leads to 6 sialyltransferase and CMP-[14C]NeuAc, only 61% of the galactose incorporated [14C]NeuAc. From the known specificites of these enzymes, it is concluded that galactose residues important in lectin binding are present in a terminal Gal/beta1 leads to 4GlcNAc sequence on asialo-factor VIII/von Willebrand factor. The relative ristocetin-induced platelet aggregating activity of native, asialo-, and agalacto-factor VIII/von Willebrand factor was 100:38:12, respectively, while procoagulant activity was 100:100:103. (+info)
Tandem amino acid repeats from Trypanosoma cruzi shed antigens increase the half-life of proteins in blood.
Proteins containing amino acid repeats are widespread among protozoan parasites. It has been suggested that these repetitive structures act as immunomodulators, but other functional aspects may be of primary importance. We have recently suggested that tandem repeats present in Trypanosoma cruzi trans-sialidase stabilize the catalytic activity in blood. Because the parasite releases trans-sialidase, this delayed clearance of the enzyme might have implications in vivo. In the present work, the ability of repetitive units from different T. cruzi molecules in stabilizing trans-sialidase activity in blood was evaluated. It is shown that repeats present on T. cruzi shed proteins (antigens 13 and Shed-Acute-Phase-Antigen [SAPA]) increase trans-sialidase half-life in blood from 7 to almost 35 hours. Conversely, those repeats present in intracellular T. cruzi proteins only increase the enzyme half-life in blood up to 15 hours. Despite these results, comparative analysis of structural and catalytic properties of both groups of chimeric enzymes show no substantial differences. Interestingly, antigens 13 and SAPA also increase the persistence in blood of chimeric glutathione S-transferases, thus suggesting that this effect is inherent to these repeats and independent of the carrier protein. Although the molecular basis of this phenomenon is still uncertain, its biotechnological potential can be envisaged. (+info)
Amino acid substitutions in a conserved region in the stalk of the Newcastle disease virus HN glycoprotein spike impair its neuraminidase activity in the globular domain.
The ectodomain of the paramyxovirus haemagglutinin-neuraminidase (HN) glycoprotein spike can be divided into two regions: a membrane-proximal, stalk-like structure and a terminal globular domain. The latter contains all the antibody recognition sites of the protein, as well as its receptor recognition and neuraminidase (NA) active sites. These two activities of the protein can be separated by monoclonal antibody functional inhibition studies and mutations in the globular domain. Herein, we show that mutation of several conserved residues in the stalk of the Newcastle disease virus HN protein markedly decrease its NA activity without a significant effect on receptor recognition. Thus, mutations in the stalk, distant from the NA active site in the globular domain, can also separate attachment and NA. These results add to an increasing body of evidence that the NA activity of this protein is dependent on an intact stalk structure. (+info)
Ortho- and paramyxoviruses from migrating feral ducks: characterization of a new group of influenza A viruses.
Ortho- and parainfluenza viruses isolated from the cloacas of migrating feral ducks shot on the Mississippi flyway included three strains of influenza. A virus (Hav6 Nav1, Hav6 Nl, Hav7 Neq2) as well as Newcastle disease virus. One influenza virus, A/duck/Memphis/546/74, possessed Hav3 haemagglutinin, but the neuraminidase was not inhibited by any of the known influenza reference antisera. The neuraminidase on this virus was related to the neuraminidases on A/duck/GDR/72 (H2 N?), A/turkey/Ontario/7732/66 (Hav 5 N?), A/duck/Ukraine/1/60 (Hav3 N?) and A/turkey/Wisconsin/68. We therefore propose that the neuraminidase on this group of influenza viruses be designated Nav6. The A/duck/Memphis/546/74 influenza virus caused an ocular discharge in 1 of 5 ducks and was shed in faeces for 10 days; it was stable in faecal samples for up to 3 days at 20 degrees C. These results suggest that ecological studies on influenza in avian species should include attempts to isolate virus from faeces. Faecal-oral transmission is an attractive explanation for the spread of influenza virus from feral birds to other animals. (+info)