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Site-directed removal of N-glycosylation sites in the bovine cation-dependent mannose 6-phosphate receptor: effects on ligand binding, intracellular targetting and association with binding immunoglobulin protein.

ABSTRACT: The bovine cation-dependent mannose 6-phosphate receptor (CD-MPR) contains five potential N-linked glycosylation sites, four of which are utilized. To evaluate the function of these oligosaccharides, site-directed mutagenesis was used to generate glycosylation-deficient CD-MPR mutants lacking various potential glycosylation sites. The mutants were constructed in both a full-length and a soluble truncated (STOP155 construct) form of the receptor and their properties were examined in transfected COS-1 cells. The results showed that the presence of a single oligosaccharide chain, particularly at position 87, on the CD-MPR significantly enhanced its mannose 6-phosphate (Man-6-P)-binding ability when compared with non-glycosylated receptors. In addition, the presence of a single oligosaccharide chain at position 87, and to a lesser degree at position 31 or 81, promoted the secretion of the STOP155 CD-MPR. Pulse-labelling of transfected COS-1 cells followed by immunoprecipitation with binding immunoglobulin protein (BiP)-specific and CD-MPR-specific antibodies indicated that BiP associated with the non-glycosylated forms of the receptor but not with the wild-type CD-MPR. Furthermore, the association of the various glycosylation-deficient forms of the CD-MPR with BiP correlated inversely with their ability to bind Man-6-P. From these results we conclude that N-glycosylation of the bovine CD-MPR facilities the folding of the nascent polypeptide chain into a conformation that is conductive for intracellular transport and ligand binding.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC1134638 | biostudies-other | 1993 Nov

REPOSITORIES: biostudies-other

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Cation-independent mannose 6-phosphate receptor: a composite of distinct phosphomannosyl binding sites.

Project description:The 300-kDa cation-independent mannose 6-phosphate receptor (CI-MPR), which contains multiple mannose 6-phosphate (Man-6-P) binding sites that map to domains 3, 5, and 9 within its 15-domain extracytoplasmic region, functions as an efficient carrier of Man-6-P-containing lysosomal enzymes. To determine the types of phosphorylated N-glycans recognized by each of the three carbohydrate binding sites of the CI-MPR, a phosphorylated glycan microarray was probed with truncated forms of the CI-MPR. Surface plasmon resonance analyses using lysosomal enzymes with defined N-glycans were performed to evaluate whether multiple domains are needed to form a stable, high affinity carbohydrate binding pocket. Like domain 3, adjacent domains increase the affinity of domain 5 for phosphomannosyl residues, with domain 5 exhibiting approximately 60-fold higher affinity for lysosomal enzymes containing the phosphodiester Man-P-GlcNAc when in the context of a construct encoding domains 5-9. In contrast, domain 9 does not require additional domains for high affinity binding. The three sites differ in their glycan specificity, with only domain 5 being capable of recognizing Man-P-GlcNAc. In addition, domain 9, unlike domains 1-3, interacts with Man(8)GlcNAc(2) and Man(9)GlcNAc(2) oligosaccharides containing a single phosphomonoester. Together, these data indicate that the assembly of three unique carbohydrate binding sites allows the CI-MPR to interact with the structurally diverse phosphorylated N-glycans it encounters on newly synthesized lysosomal enzymes.

| S-EPMC2787381 | biostudies-literature

Site-directed removal of N-glycosylation sites in BST-1/CD157: effects on molecular and functional heterogeneity.

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Structural insights into the mechanism of pH-dependent ligand binding and release by the cation-dependent mannose 6-phosphate receptor.

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Ion- and water-binding sites inside an occluded hourglass pore of a trimeric intracellular cation (TRIC) channel.

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Identification of a fourth mannose 6-phosphate binding site in the cation-independent mannose 6-phosphate receptor.

Project description:The 300 kDa cation-independent mannose 6-phosphate receptor (CI-MPR) plays an essential role in lysosome biogenesis by targeting ? 60 different phosphomannosyl-containing acid hydrolases to the lysosome. This type I membrane glycoprotein has a large extracellular region comprised of 15 homologous domains. Two mannose 6-phosphate (M6P) binding sites have been mapped to domains 3 and 9, whereas domain 5 binds preferentially to the phosphodiester, M6P-N-acetylglucosamine (GlcNAc). A structure-based sequence alignment predicts that the C-terminal domain 15 contains three out of the four conserved residues identified as essential for carbohydrate recognition by domains 3, 5 and 9 of the CI-MPR, but lacks two cysteine residues that are predicted to form a disulfide bond. To determine whether domain 15 of the CI-MPR has lectin activity and to probe its carbohydrate-binding specificity, truncated forms of the CI-MPR were tested for binding to acid hydrolases with defined N-glycans in surface plasmon resonance analyses, and used to interrogate a phosphorylated glycan microarray. The results show that a construct encoding domains 14-15 binds both M6P and M6P-GlcNAc with similar affinity (Kd = 13 and 17 ?M, respectively). Site-directed mutagenesis studies demonstrate the essential role of the conserved Tyr residue in domain 15 for phosphomannosyl binding. A structural model of domain 15 was generated that predicted an Arg residue to be in the binding pocket and mutagenesis studies confirmed its important role in carbohydrate binding. Together, these results show that the CI-MPR contains a fourth carbohydrate-recognition site capable of binding both phosphomonoesters and phosphodiesters.

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The effects of the site-directed removal of N-glycosylation sites from beta-1,4-N-acetylgalactosaminyltransferase on its function.

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Ligand-binding characteristics of feline insulin-binding immunoglobulin G.

Project description:Polyclonal immunoglobulin (Ig) G autoantibodies against insulin have been identified in sera of healthy cats. We purified and fractionated insulin-binding IgGs from cat sera by affinity chromatography and analyzed affinity of insulin-binding IgGs for insulin and their epitopes. Following the passing of fraction A, which did not bind to insulin, insulin-binding IgGs were eluted into two fractions, B and C, by affinity chromatography using a column fixed with bovine insulin. Dissociation constant (KD) values between insulin-binding IgGs and insulin, determined by surface plasmon resonance analysis (Biacore™system), were 1.64e(-4) M for fraction B (low affinity IgGs) and 2e(-5) M for fraction C (high affinity IgGs). Epitope analysis was conducted using 16 peptide fragments synthesized in concord with the amino acid sequence of feline insulin by an enzyme-linked immunosorbent assay. Fractions B and C showed higher absorbance (affinity) of the peptide fragment of 10 amino acid residues at the carboxyl-terminal of the B chain (peptide No. 19), followed by peptide fragments of 6 to 15 amino acid residues of the B chain (peptide No. 8). Fraction C showed a higher absorbance to 7 to 16 amino acid residues of the B chain (peptide No. 5) compared with the absorbance of fraction B. Polyclonal insulin-binding IgGs may form a macromolecule complex with insulin through the multiple affinity sites of IgG molecules. Feline insulin-binding IgGs are multifocal and may be composed of multiple IgG components and insulin.

| S-EPMC4667653 | biostudies-literature

Residues essential for plasminogen binding by the cation-independent mannose 6-phosphate receptor.

Project description:The 300 kDa cation-independent mannose 6-phosphate receptor (CI-MPR) is a multifunctional protein that binds diverse intracellular and extracellular ligands with high affinity. The CI-MPR is a receptor for plasminogen, and this interaction can be inhibited by lysine analogues. To characterize the molecular basis for this interaction, surface plasmon resonance (SPR) analyses were performed using truncated forms of the CI-MPR and plasminogen. The results show that the N-terminal region of the CI-MPR containing domains 1 and 2, but not domain 1 alone, of the receptor's 15-domain extracytoplasmic region binds plasminogen (K(d) = 5 +/- 1 nM) with an affinity similar to that of the full-length receptor (K(d) = 20 +/- 6 nM). In addition to its C-terminal serine protease domain, plasminogen contains lysine binding sites (LBS), which are located within each of its five kringle domains, except kringle 3. We show that kringles 1-4, but not kringles 1-3, bind the CI-MPR, indicating an essential role for the LBS in kringle 4 of plasminogen. To identify the lysine residue(s) of the CI-MPR that serve(s) as an essential determinant for recognition by the LBS of plasminogen, site-directed mutagenesis studies were carried out using a construct encoding the N-terminal three domains of the CI-MPR (Dom1-3His) which contains both a mannose 6-phosphate (Man-6-P) and plasminogen binding site. The results demonstrate two lysine residues (Lys53 located in domain 1 and Lys125 located in the loop connecting domains 1 and 2) of the CI-MPR are key determinants for plasminogen binding but are not required for Man-6-P binding.

| S-EPMC2814163 | biostudies-literature

Tissue plasminogen activator is a ligand of cation-independent mannose 6-phosphate receptor and consists of glycoforms that contain mannose 6-phosphate.

Project description:Plasmin is the key enzyme in fibrinolysis. Upon interaction with plasminogen activators, the zymogen plasminogen is converted to active plasmin. Some studies indicate plasminogen activation is regulated by cation-independent mannose 6-phosphate receptor (CI-MPR), a protein that facilitates lysosomal enzyme trafficking and insulin-like growth factor 2 downregulation. Plasminogen regulation may be accomplished by CI-MPR binding to plasminogen or urokinase plasminogen activator receptor. We asked whether other members of the plasminogen activation system, such as tissue plasminogen activator (tPA), also interact with CI-MPR. Because tPA is a glycoprotein with three N-linked glycosylation sites, we hypothesized that tPA contains mannose 6-phosphate (M6P) and binds CI-MPR in a M6P-dependent manner. Using surface plasmon resonance, we found that two sources of tPA bound the extracellular region of human and bovine CI-MPR with low-mid nanomolar affinities. Binding was partially inhibited with phosphatase treatment or M6P. Subsequent studies revealed that the five N-terminal domains of CI-MPR were sufficient for tPA binding, and this interaction was also partially mediated by M6P. The three glycosylation sites of tPA were analyzed by mass spectrometry, and glycoforms containing M6P and M6P-N-acetylglucosamine were identified at position N448 of tPA. In summary, we found that tPA contains M6P and is a CI-MPR ligand.

| S-EPMC8050316 | biostudies-literature

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