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Characterization of the glycosyl-phosphatidylinositol-anchored human renal dipeptidase reveals that it is more extensively glycosylated than the pig enzyme.

ABSTRACT: Renal dipeptidase (EC 3.4.13.11) has been purified from human kidney cortex by affinity chromatography on cilastatin-Sepharose following solubilization with either n-octyl-beta-D-glucopyranoside or bacterial phosphatidylinositol-specific phospholipase C (PI-PLC). Phase separation in Triton X-114 revealed that the detergent-solubilized form was amphipathic and retained the glycosyl-phosphatidylinositol membrane anchor whereas the phospholipase solubilized form was hydrophilic. Both forms of the enzyme existed as a disulphide-linked dimer of two identical subunits of Mr 59,000 each. The glycosyl-phosphatidylinositol anchor of purified human renal dipeptidase was hydrolysed by a range of bacterial PI-PLCs and by a plasma phospholipase D. Mild acid treatment and nitrous acid deamination of the hydrophilic form revealed that the cross-reacting determinant, characteristic of the glycosyl-phosphatidylinositol anchor, was due exclusively to the inositol 1,2-cyclic phosphate ring epitope. The N-terminal amino acid sequences of the amphipathic and hydrophilic forms were identical, locating the membrane anchor at the C-terminus. The N-terminal sequence of human renal dipeptidase showed a high degree of similarity with that of the pig enzyme, and enzymic deglycosylation revealed that the difference in size of renal dipeptidase between these two species is due almost entirely to differences in the extent of N-linked glycosylation.

SUBMITTER: Hooper NM

PROVIDER: S-EPMC1136904 | biostudies-other | 1990 Jan

REPOSITORIES: biostudies-other

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cDNA cloning and expression in Xenopus laevis oocytes of pig renal dipeptidase, a glycosyl-phosphatidylinositol-anchored ectoenzyme.

Project description:Clones expressing renal dipeptidase (EC 3.4.13.11) have been isolated from a pig kidney cortex cDNA library after employing the polymerase chain reaction technique to amplify a region of the dipeptidase cDNA. The complete primary sequence of the enzyme has been deduced from a full length cDNA clone. This predicts a protein of 409 amino acids, a cleavable N-terminal signal sequence of 16 residues and two N-linked glycosylation sites. At the C-terminus of the predicted sequence is a stretch of mainly hydrophobic amino acids which is presumed to direct the attachment of the glycosyl-phosphatidylinositol membrane anchor. Expression of the mRNA for pig renal dipeptidase in Xenopus laevis oocytes led to the production of a disulphide-linked dimeric protein of subunit Mr 48,600 which was recognized by a polyclonal antiserum raised to renal dipeptidase purified from pig kidney cortex. Bacterial phosphatidylinositol-specific phospholipase C released renal dipeptidase from the surface of the oocytes and converted the amphipathic detergent-solubilized form of the dipeptidase to a hydrophilic form, indicating that Xenopus laevis oocytes can process expressed proteins to their glycosyl-phosphatidylinositol anchored form.

| S-EPMC1149627 | biostudies-other

Activation of the glycosyl-phosphatidylinositol-anchored membrane dipeptidase upon release from pig kidney membranes by phospholipase C.

Project description:Incubation of pig kidney microvillar membranes with Bacillus thuringiensis or Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) resulted in the release of a number of glycosyl-phosphatidylinositol (GPI)-anchored hydrolases, including alkaline phosphatase (EC 3.1.3.1), amino-peptidase P (EC 3.4.11.9), membrane dipeptidase (EC 3.4.13.19), 5'-nucleotidase (EC 3.1.3.5) and trehalase (EC 3.2.1.28). Of these five ectoenzymes only for membrane dipeptidase was there a significant (approx. 100%) increase in enzymic activity upon release from the membrane. Maximal activation occurred at a PI-PLC concentration 10-fold less than that required for maximal release. In contrast solubilization of the membranes with n-octyl beta-D-glucopyranoside had no effect on the enzymic activity of membrane dipeptidase. A competitive e.l.i.s.a. with a polyclonal antiserum to membrane dipeptidase indicated that the increase in enzymic activity was not due to an increase in the amount of membrane dipeptidase protein. Although PI-PLC cleaved the GPI anchor of the affinity-purified amphipathic form of pig membrane dipeptidase there was no concurrent increase in enzymic activity. In the absence of PI-PLC, membrane dipeptidase in the microvillar membranes hydrolysed Gly-D-Phe with a Km of 0.77 mM and a Vmax. of 602 nmol/min per mg of protein. However, in the presence of a concentration of PI-PLC which caused maximal release from the membrane and maximal activation of membrane dipeptidase the Km was decreased to 0.07 mM while the Vmax. remained essentially unchanged at 624 nmol/min per mg of protein. Overall these results suggest that cleavage by PI-PLC of the GPI anchor on membrane dipeptidase may relax conformational constraints on the active site of the enzyme which exist when it is anchored in the lipid bilayer, thus resulting in an increase in the affinity of the active site for substrate.

| S-EPMC1137374 | biostudies-other

Glycosyl phosphatidylinositol-anchored proteins in chemosensory signaling: antisense manipulation of Paramecium tetraurelia PIG-A gene expression.

Project description:Glycosyl phosphatidylinositol (GPI)-anchored proteins are peripheral membrane proteins tethered to the cell through a lipid anchor. GPI-anchored proteins serve many functions in cellular physiology and cell signaling. The PIG-A gene codes for one of the enzymes of a complex that catalyzes the first step in anchor synthesis, and we have cloned the Paramecium tetraurelia pPIG-A gene using homology PCR. To understand the function of pPIG-A and the significance of GPI-anchored proteins in Paramecium, we reduced the mRNA for pPIG-A in transformed cells using an expression vector that transcribed antisense mRNA. The amount of transcript is reduced to approximately 0.3% of the mRNA in control-transformed cells. Compared to control cells, cells transformed with the antisense pPIG-A vector show reduced synthesis of GPI anchor intermediates catalyzed in their endoplasmic reticula and a very few GPI-anchored proteins among the peripheral proteins that can be recovered from their surfaces. They also show specific defects in chemoresponse to glutamate and folate. Other cellular functions, such as growth and mating, seem to be normal.

| S-EPMC326658 | biostudies-literature

Purification and characterization of pig kidney aminopeptidase P. A glycosyl-phosphatidylinositol-anchored ectoenzyme.

Project description:Aminopeptidase P (EC 3.4.11.9) was solubilized from pig kidney membranes with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) and then purified by a combination of anion-exchange and hydrophobic-interaction chromatographies. Contaminating peptidase activities were removed by selective affinity chromatography. The purified enzyme was apparently homogeneous on SDS/PAGE with an Mr of 91,000. Enzymic deglycosylation revealed that aminopeptidase P is a glycoprotein, with up to 25% by weight of the protein being due to the presence of N-linked sugars. The phospholipase-solubilized aminopeptidase P was recognized by an antiserum to the cross-reacting determinant (CRD) characteristic of the glycosyl-phosphatidylinositol anchor. This recognition was abolished by mild acid treatment or deamination with HNO2, indicating that the CRD was due exclusively to the inositol 1,2-cyclic phosphate ring epitope generated by the action of PI-PLC. The activity of aminopeptidase P was inhibited by chelating agents and was stimulated by Mn2+ or Co2+ ions, confirming the metallo-enzyme nature of this peptidase. Selective inhibitors of other aminopeptidases (actinonin, amastatin, bestatin and puromycin) had little or no inhibitory effect.

| S-EPMC1131318 | biostudies-other

Insulin stimulates the release of the glycosyl phosphatidylinositol-anchored membrane dipeptidase from 3T3-L1 adipocytes through the action of a phospholipase C.

Project description:Membrane dipeptidase (MDP; EC 3.4.13.19) enzymic activity that was inhibited by cilastatin has been detected on the surface of 3T3-L1 cells. On differentiation of the cells from fibroblasts to adipocytes the activity of MDP increased 12-fold. Immunoelectrophoretic blot analysis indicated that on adipogenesis the increase in the amount of MDP preceded the appearance of GLUT-4. MDP on 3T3-L1 adipocytes was anchored in the bilayer by a glycosyl phosphatidylinositol (GPI) moiety as evidenced by its release into the medium in a hydrophilic form on treatment of the cells with bacterial phosphatidylinositol-specific phospholipase C and the appearance of the inositol 1,2-cyclic monophosphate cross-reacting determinant. Incubation of 3T3-L1 adipocytes with either insulin or the sulphonylurea glimepiride led to a rapid concentration- and time-dependent release of MDP from the cell surface. The hydrophilic form of MDP released from the cells on stimulation with insulin was recognized by antibodies against the inositol 1,2-cyclic monophosphate cross-reacting determinant, indicating that it had been generated by cleavage of its GPI anchor through the action of a phospholipase C.

| S-EPMC1218701 | biostudies-other

Identification of membrane dipeptidase as a major glycosyl-phosphatidylinositol-anchored protein of the pancreatic zymogen granule membrane, and evidence for its release by phospholipase A.

Project description:Membrane dipeptidase (EC 3.4.13.19) enzyme activity that is inhibited by cilastatin has been detected in pancreatic zymogen granule membranes of human, porcine and rat origin. Immunoelectrophoretic blot analysis of human and porcine pancreatic zymogen granule membranes with polyclonal antisera raised against the corresponding kidney membrane dipeptidase revealed that the enzyme is a disulphide-linked homodimer of subunit mass 61 kDa in the human and 45 kDa in the pig. Although membrane dipeptidase was, along with glycoprotein-2, one of the only two major components of carbonate high pH-washed membranes, no enzyme activity or immunoreactivity was detected in the zymogen granule contents. Digestion with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), and subsequent recognition by antibodies specific for the cross-reacting determinant, revealed that membrane dipeptidase in human and porcine pancreatic zymogen granule membranes is glycosyl-phosphatidylinositol-anchored. Membrane dipeptidase was released from the pancreatic zymogen granule membranes by an endogenous hydrolase, and the released form migrated as a disulphide-linked dimer on SDS/PAGE under non-reducing conditions. Under reducing conditions it migrated with the same apparent molecular mass as the membrane-bound form, and was still a substrate for bacterial PI-PLC. Treatment of kidney microvillar membranes with phospholipase A2 resulted in the release of membrane dipeptidase in a form that demonstrated electrophoretic and cilastatin-Sepharose binding properties identical to those of the endogenously released form of the enzyme from zymogen granule membranes. These results indicate that the glycosyl-phosphatidylinositol anchor on the pancreatic membrane dipeptidase is cleaved by an endogenous hydrolase, probably a phospholipase A, and that this cleavage may promote the release of the protein from the membrane.

| S-EPMC1218411 | biostudies-other

Cytodifferentiation in Tetrahymena vorax is linked to glycosyl-phosphatidylinositol-anchored protein assembly.

Project description:The role of glycosyl-PtdIns (GPI)-anchored proteins in the cytodifferentiation of Tetrahymena vorax was examined. Labelling of cells with [3H]myristate or [3H]palmitate followed by electrophoresis showed an array of proteins carrying covalently bound lipids. Electrophoresis of protein from cells labelled with the GPI-anchor components [3H]Ins and [14C]ethanolamine revealed three polypeptides on fluorograms which have apparent molecular masses of approx. 28, 50 and 82 kDa. Labelled lipid associated with these polypeptides was susceptible to release by in vitro exposure to Bacillus thuringiensis PtdIns-specific phospholipase C (PI-PLC). Using labelled fatty acids, cells induced to differentiate showed altered GPI-anchored protein-labelling patterns in comparison with undifferentiated control cells, with a heavily labelled 32 kDa band appearing upon differentiation. Pre-incubation of cells in 10 mM D-mannosamine, an inhibitor of GPI incorporation into protein, resulted in a reduction of the incorporation of label into the three GPI-anchored proteins, nearly complete inhibition of differentiation and a reduction in the rate of digestive vacuole formation. A 50% inhibition of differentiation was obtained using 500 microM mannosamine. The inhibitory impact of D-mannosamine on differentiation could be competitively and completely reversed by the inclusion of D-mannose, but not D-glucose. Neither glucosamine nor tunicamycin inhibited differentiation. Incubation of cells in PI-PLC (5 units/ml) plus the differentiation inducer resulted in an acceleration of differentiation and generally higher percentages of differentiated cells versus controls.

| S-EPMC1137916 | biostudies-other

Dynamic partitioning of a glycosyl-phosphatidylinositol-anchored protein in glycosphingolipid-rich microdomains imaged by single-quantum dot tracking.

Project description:Recent experimental developments have led to a revision of the classical fluid mosaic model proposed by Singer and Nicholson more than 35 years ago. In particular, it is now well established that lipids and proteins diffuse heterogeneously in cell plasma membranes. Their complex motion patterns reflect the dynamic structure and composition of the membrane itself, as well as the presence of the underlying cytoskeleton scaffold and that of the extracellular matrix. How the structural organization of plasma membranes influences the diffusion of individual proteins remains a challenging, yet central, question for cell signaling and its regulation. Here we have developed a raft-associated glycosyl-phosphatidyl-inositol-anchored avidin test probe (Av-GPI), whose diffusion patterns indirectly report on the structure and dynamics of putative raft microdomains in the membrane of HeLa cells. Labeling with quantum dots (qdots) allowed high-resolution and long-term tracking of individual Av-GPI and the classification of their various diffusive behaviors. Using dual-color total internal reflection fluorescence (TIRF) microscopy, we studied the correlation between the diffusion of individual Av-GPI and the location of glycosphingolipid GM1-rich microdomains and caveolae. We show that Av-GPI exhibit a fast and a slow diffusion regime in different membrane regions, and that slowing down of their diffusion is correlated with entry in GM1-rich microdomains located in close proximity to, but distinct, from caveolae. We further show that Av-GPI dynamically partition in and out of these microdomains in a cholesterol-dependent manner. Our results provide direct evidence that cholesterol-/sphingolipid-rich microdomains can compartmentalize the diffusion of GPI-anchored proteins in living cells and that the dynamic partitioning raft model appropriately describes the diffusive behavior of some raft-associated proteins across the plasma membrane.

| S-EPMC2766537 | biostudies-literature

Transient anchorage of cross-linked glycosyl-phosphatidylinositol-anchored proteins depends on cholesterol, Src family kinases, caveolin, and phosphoinositides.

Project description:How outer leaflet plasma membrane components, including glycosyl-phosphatidylinositol-anchored proteins (GPIAPs), transmit signals to the cell interior is an open question in membrane biology. By deliberately cross-linking several GPIAPs under antibody-conjugated 40-nm gold particles, transient anchorage of the gold particle-induced clusters of both Thy-1 and CD73, a 5' exonucleotidase, occurred for periods ranging from 300 ms to 10 s in fibroblasts. Transient anchorage was abolished by cholesterol depletion, addition of the Src family kinase (SFK) inhibitor PP2, or in Src-Yes-Fyn knockout cells. Caveolin-1 knockout cells exhibited a reduced transient anchorage time, suggesting the partial participation of caveolin-1. In contrast, a transmembrane protein, the cystic fibrosis transmembrane conductance regulator, exhibited transient anchorage that occurred without deliberately enhanced cross-linking; moreover, it was only slightly inhibited by cholesterol depletion or SFK inhibition and depended completely on the interaction of its PDZ-binding domain with the cytoskeletal adaptor EBP50. We propose that cross-linked GPIAPs become transiently anchored via a cholesterol-dependent SFK-regulatable linkage between a transmembrane cluster sensor and the cytoskeleton.

| S-EPMC2064508 | biostudies-literature

Recombinant glycosyl-phosphatidylinositol-anchored proteins are not associated with protein kinases in transfected thymoma cells.

Project description:The cross-linking by antibody of some glycosyl-phosphatidyl-inositol (GPI)-anchored proteins on the plasma membrane of T cells leads to cell activation. Phosphorylation of proteins on tyrosine residues has a central role in the control of T cell activation, and non-receptor protein tyrosine kinases can be coprecipitated with immune complexes of GPI-anchored proteins in T cell lysates. In order to investigate the nature of this interaction, two recombinant GPI-anchored proteins were constructed (using the GPI signal sequence from Thy-1), and their associations with protein tyrosine kinases in stable transfectants of a mouse thymoma have been investigated. One recombinant GPI protein is the extracellular domain of the human complement receptor-1, normally an integral membrane protein, and the other is the secreted protein, human tissue inhibitor of metalloproteinases. The latter protein should be foreign to the cell surface and yet has been expressed as a GPI-anchored protein at levels equivalent to the highly expressed antigens Thy-1 and Ly6.A2 on mouse thymoma cells. Neither of the two recombinant proteins, when immunoprecipitated from NP40 lysates of transfected cells, was associated with protein tyrosine kinases in contrast with the natural endogenous GPI-anchored proteins Thy-1 and Ly6.A2 in non-transfected parental cells. Moreover, high expression of foreign recombinant GPI protein appears to interfere with the association of the natural GPI proteins with protein tyrosine kinases.

| S-EPMC1137412 | biostudies-other

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