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

ABSTRACT: 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.

SUBMITTER: Hooper NM

PROVIDER: S-EPMC1131318 | biostudies-other | 1990 Apr

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

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

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.

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

Project description: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.

| S-EPMC1136904 | biostudies-other

Proteins of the kidney microvillar membrane. The 130 kDa protein in pig kidney, recognized by monoclonal antibody GK5C1, is an ectoenzyme with aminopeptidase activity.

Project description:The hybridoma GK5C1, secreting a monoclonal IgG1 antibody, was generated after immunizing a mouse with pig kidney microvillar membranes. An immunoradiometric assay showed that only kidney and intestine contained detectable amounts of the antigen recognized by the antibody, the highest concentration being observed in the ileum. Immunocytochemistry confirmed this observation and revealed that the antigen was associated with renal and intestinal brush borders. By 'Western' blotting, the antigen in kidney microvilli was shown to be a 130 kDa polypeptide. Papain treatment of the membrane before blotting converted the antigen to a 125 kDa polypeptide, no longer associated with membrane. Immunoaffinity chromatography of detergent-solubilized kidney membranes yielded a pure 130 kDa protein. When one purification was monitored by the immunoradiometric assay, the yield was 3.5% and the purification factor was 1000-fold. The antigen constituted about 0.8% of the microvillar membrane protein. The protein could be reconstituted into liposomes, where electron microscopy revealed an asymmetric orientation, similar to that of ectoenzymes in this membrane. The stalk length was about 3 nm. In electron micrographs the purified protein appeared to be dimeric. A search for enzymic activity was rewarded when L-leucyl-L-tryptophan was observed to be hydrolysed. Failure to hydrolyse N-blocked peptides and the ability to release the N-terminal residue from extended peptides, including Leu-Trp-Leu and Leu-Trp-Met-Arg, showed that the activity was that of an aminopeptidase. The enzyme was maximally active at pH 7.5 and irreversibly inactivated outside the range pH 6-10. This activity could not be attributed to trace contamination with aminopeptidase N. The best substrates so far identified for the 130 kDa protein were those with tryptophan in the P1', position. This protein is a new microvillar enzyme and it is proposed that it be called aminopeptidase W.

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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.

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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

Soluble low-Km 5'-nucleotidase from electric-ray (Torpedo marmorata) electric organ and bovine cerebral cortex is derived from the glycosyl-phosphatidylinositol-anchored ectoenzyme by phospholipase C cleavage.

Project description:Soluble and membrane-bound low-Km 5'-nucleotidase was isolated from high-speed supernatants and membrane fractions derived from the electric organ of the electric ray (Torpedo marmorata) or from bovine brain cerebral cortex. Purification of both enzymes included chromatography on concanavalin A-Sepharose and AMP-Sepharose. The contribution to the total of soluble enzyme activity was lower in electric organ (1.6%) than in bovine cerebral cortex (27.9%). Membrane-bound and soluble forms have very similar Km values for AMP and are inhibited by micromolar concentrations of ATP. Both forms cross-react with, and are inhibited by, an antibody against the membrane-bound surface-located (ecto-) 5'-nucleotidase from electric organ. The HNK-1 carbohydrate epitope is present on both forms of the Torpedo enzyme, but is entirely absent from bovine cerebral-cortex 5'-nucleotidase. An antibody specific for the inositol 1,2-(cyclic)monophosphate that is formed on phospholipase C cleavage of an intact glycosyl-phosphatidylinositol (GPI) anchor binds to the soluble, but not to the membrane-bound, form of the enzyme from both sources. Our results suggest that soluble low-Km 5'-nucleotidase in both electric organ and bovine brain is derived from the membrane-bound GPI-anchored form of the enzyme by the action of a phospholipase C and is not a soluble cytoplasmic enzyme.

| S-EPMC1132579 | biostudies-other

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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