HCO3(-)-dependent conformational change in gastric parietal cell AE2, a glycoprotein naturally lacking sialic acid

1996 ◽  
Vol 271 (2) ◽  
pp. G311-G321 ◽  
Author(s):  
A. S. Zolotarev ◽  
R. R. Townsend ◽  
A. Stuart-Tilley ◽  
S. L. Alper

Although the AE1 chloride/bicarbonate exchanger of the red blood cell is among the most thoroughly investigated of membrane transport proteins, less is known about the related AE2 polypeptide of parietal cells. We have studied enzymatic deglycosylation of native AE2 polypeptide in gastric mucosal membranes from pig and rabbit. Deglycosylation of AE2 was maximal at low ionic strength. Deglycosylation of AE2 in membranes was preferentially inhibited by bicarbonate compared with other anions. This inhibition was maximal at alkaline pH and was not evident after detergent solubilization of AE2. Deglycosylation of AE2 increased its susceptibility to proteolytic degradation, but the presence of bicarbonate protected against this degradation. Bicarbonate failed to inhibit deglycosylation of the membrane glycoproteins AE1 and gastric H(+)-K(+)-adenosinetriphosphatase beta-subunit or deglycosylation of the soluble glycoproteins fetuin and ribonuclease B. These data suggest that bicarbonate induces a conformational change in AE2 that can protect the polypeptide from deglycosylation and proteolysis. Pig AE2 was purified in sodium dodecyl sulfate, and its monosaccharide composition was determined after blotting onto polyvinylidene fluoride membrane. AE2 was found to be devoid of sialic acid, with a composition suggestive of the presence of lactosamine-type chains.

1981 ◽  
Author(s):  
B Toor ◽  
J L McGregor ◽  
K J Clemetson ◽  
L McGregor ◽  
M Dechavanne ◽  
...  

Rabbit and rat platelets have been extensively investigated under in vitro or in vivo conditions to try to understand the pathology of thrombosis in man. Here, surface-labelling techniques have been used to find out if the platelet surface has a similar composition in these two animals and in man or not. Human, rabbit and rat platelets were isolated, washed and surface-labelled by techniques specific for protein or for sugars (sialic acid or penultimate galactose/N-acetyl galactosamine residues). Labelled platelets were solubilized in sodium dodecyl sulphate and separated under reducing conditions on 7.5 % Laemmli polyacrylamide gels. Dried gels were exposed to film by fluorography or indirect autoradiography. Terminal Gal/Gal NAc residues (no neuraminidase treatment) were strongly labelled with rat and rabbit platelets compared to human platelets which labelled very poorly. Terminal sialic acid labelling with rat and rabbit platelets showed a weak labelling of a glycoprotein (GP) with the same M.Wt. as GPIb which is the most intensely labelled GP in man. However two GP (with rabbits) and one GP (in rats) were intensely labelled at a M.Wt. similar to that of GPIa in man. These GP had a different M.Wt. with terminal Gal/Gal NAc labelling. Bands with a similar M.Wt. to GPIIb and IIIa in man were strongly iodinated with rabbit platelets but with rat platelets only a single band at the position of GPIIb was strongly iodinated. These results strongly indicate that there are considerable differences in surface composition between rabbit, rat and human platelets.


1979 ◽  
Vol 42 (05) ◽  
pp. 1490-1502 ◽  
Author(s):  
C S P Jenkins ◽  
E F Ali-Briggs ◽  
G T E Zonneveld ◽  
A Sturk ◽  
J Clemetson

SummaryThe separation of the major platelet membrane glycoproteins of normal subjects and subjects with well defined platelet membrane glycoprotein abnormalities have been examined using four different polyacrylamide gel electrophoretic techniques (continuous and discontinuous). The mobilities of the resolved glycoprotein bands have been correlated with the glycoprotein nomenclature proposed for the conventional sodium dodecyl sulphate- phosphate buffer system. Since the glycoprotein distribution varies depending on the system used, the merits of each method should be considered before application to a specific problem.


1982 ◽  
Vol 207 (1) ◽  
pp. 37-41 ◽  
Author(s):  
A Imam ◽  
D J Laurence ◽  
A M Neville

Two individual glycoprotein components from human milk-fat-globule membranes (MFGM) has been purified by selectively extracting the membrane glycoproteins followed by lectin affinity chromatography and gel filtration on Sephadex G-200 in the presence of protein-disaggregating agents. The purified glycoprotein components, termed ‘epithelial-membrane glycoprotein’ (EMGP-155 and EMGP-39) have estimated molecular weights of 155 000 and 39 000 respectively, and yield a single band under reducing conditions on sodium dodecyl sulphate/polyacrylamide gel. EMGP-155 and EMGP-39 contain 21.0% and 7.0% carbohydrate by weight, with fucose (13.5%, 12.4%), mannose (3.7%, 6.2%), galactose (28.5%, 22.6%), N-acetylglucosamine (17.8%, 7.4%) and sialic acid (36.4%, 51.4%) of the carbohydrate moiety respectively. For both the glycoprotein components, aspartic and glutamic acid and serine are the major amino acid residues.


1976 ◽  
Vol 156 (1) ◽  
pp. 143-150 ◽  
Author(s):  
R H Quarles

Rats (14 days old) were injected with [14c]fucose and young adult rats with [3H]fucose in order to label the myelin-associated glycoproteins. As previously reported, the major [14C]fucose-labelled glycoprotein in the immature myelin had a higher apparent molecular weight on sodium dodecyl sulphate/polyacrylamide gels that the [3H]fucose-labelled glycoprotein in mature myelin. This predominant doubly labelled glycoprotein component was partially purified by preparative gel electrophoresis and converted to glycopeptides by extensive Pronase digestion. Gel filtration on Sephadex G-50 separated the glycopeptides into several clases, which were designted A,B, C AND D, from high to low molecular weight. The 14C-labelled glycopeptides from immature myeline were enriched in the highest-molecular-weight class A relative to the 3H-labelled glycopeptides from mature myelin. Neuraminidase treatment of the glycoprotein before Pronase digestion greatly decreased the proportion of glycopeptides fractionating in the higher-molecular-weight classes and largely eliminated the developmental differences that were apparent by gel filtration. However, neuraminidase treatment did not decrease the magnitude of the developmental difference revealed by electrophoresing the intact glycoprotein on sodium dodecyl sulphate gels, although it did decrease the apparent molecular weight of the glycoprotein from both the 15-day-old and adult rats by an amount comparable in magnitude to that developmental difference. The results from gel filtration of glycopeptides indicate that there is a higher content of large molecular weight, sialic acid-rich oligosaccharide units in the glycoprotein of immature myelin. However, the higher apparent molecular weight for the glycoprotein from 15-day-old rats on sodium dodcyl sulphate gels is not due primarily to its higher sialic acid content.


Blood ◽  
1985 ◽  
Vol 66 (5) ◽  
pp. 1068-1071 ◽  
Author(s):  
MA Baker ◽  
RN Taub ◽  
A Kanani ◽  
I Brockhausen ◽  
A Hindenburg

Granulocytes from patients with chronic myelogenous leukemia (CML) are morphologically identical to their normal counterparts but show marked differences in circulation patterns and in some membrane properties. We have previously shown that there is abnormal lectin binding to CML granulocytes, and aberrant sialylation of membrane glycoproteins. To examine the changes in sialylation of CML granulocytes further, we have studied membrane preparations from CML and normal granulocytes for specific sialyltransferase activity. Because sialyltransferase enzymes are specific for the configuration of the acceptor group, enzyme activity was assayed by measuring transfer of sialic acid from CMP-14C- sialic acid to substrates of defined structure. As compared with those of normal counterparts, CML extracts catalyzed a 50% higher overall rate of sialylation of asialofetuin, a substrate possessing both N- and O-linked acceptors. Studies of enzyme specificity utilizing porcine and ovine submaxillary mucins, antifreeze glycoprotein and alpha-1 acid glycoprotein as acceptors showed that the increased sialylation by CML extracts was due primarily to substrates with the O-linked Gal beta 1--- -3GaINAc acceptor group. These data suggest that sialyltransferase activity is increased in CML granulocytes compared to normal granulocytes and that the increased enzyme activity is specific for O- linked Gal beta 1----3GaINAc. This enzyme activity may be directly responsible for the abnormal membrane sialylation and pathophysiological behavior of these cells.


1981 ◽  
Author(s):  
J L McGregor ◽  
K J Clemetson ◽  
E James ◽  
A Capitanio ◽  
M Dechavanne ◽  
...  

Glanzmann’s thrombasthenia (G.T.) platelets are deficient in 2 major membrane GP (IIb and IIIa). In order to investigate if these are the only defects in this disorder, platelets from G.T. patients and from healthy donors were isolated, washed and surface-labelled by techniques specific for protein or for sugars (sialic acid or penultimate galactose/N-acetylgalactosamine residues). Labelled or unlabelled platelets were solubilized in sodium dodecyl sulphate (SDS) and separated by 2-dimensional polyacrylamide gel electrophoresis, first according to isoelectric point and then according to molecular weight. Glycoproteins from unlabelled platelets separated by 2-dimensional electrophoresis were identified by binding of 125I-labelled Lens culinaris lectin (mannose, glucose specific) GPIIbA1 and IIIaA1 were absent in one G.T. patient while in others lower amounts of 2 GP were found in positions similar to these GP. Major membrane GP (IbA1, IbA2, IbB1 and IIIbA1) had more intensely labelled terminal sialic acid moieties in G.T. platelets than in normals. A major membrane GP designated Ic had an altered pi and its penultimate galactose/N-acetyl galactosamine residues labelled more intensely in G.T. platelets than in controls. One high M.Wt. GP and a number of lower M.Wt. GP (IVa, IVb and VII) normally found in platelets of healthy donors were absent in G.T. platelets. These results indicate strongly that there is a major perturbation of the platelet surface in G.T.


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