Cation-independent Mannose 6-Phosphate Receptor: A COMPOSITE OF DISTINCT PHOSPHOMANNOSYL BINDING SITES*

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 ∼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 Man₈GlcNAc₂ and Man₉GlcNAc₂ 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.     

Domain 5 of the cation-independent mannose 6-phosphate receptor preferentially binds phosphodiesters (mannose 6-phosphate N-acetylglucosamine ester)

The 300 kDa cation-independent mannose 6-phosphate receptor (CI-MPR) and the 46 kDa cation-dependent MPR (CD-MPR) are key components of the lysosomal enzyme targeting system that bind newly synthesized mannose 6-phosphate (Man-6-P)-containing acid hydrolases and divert them from the secretory pathway. Previous studies have mapped two high-affinity Man-6-P binding sites of the CI-MPR to domains 1-3 and 9 and one low-affinity site to domain 5 within its 15-domain extracytoplasmic region. A structure-based sequence alignment predicts that domain 5 contains the four conserved residues (Gln, Arg, Glu, Tyr) identified as essential for Man-6-P binding by the CD-MPR and domains 1-3 and 9 of the CI-MPR. Here we show by surface plasmon resonance (SPR) analyses of constructs containing single amino acid substitutions that these conserved residues (Gln-644, Arg-687, Glu-709, Tyr-714) are critical for carbohydrate recognition by domain 5. Furthermore, the N-glycosylation site at position 711 of domain 5, which is predicted to be located near the binding pocket, has no influence on the carbohydrate binding affinity. Endogenous ligands for the MPRs that contain solely phosphomonoesters (Man-6-P) or phosphodiesters (mannose 6-phosphate N-acetylglucosamine ester, Man-P-GlcNAc) were generated by treating the lysosomal enzyme acid alpha-glucosidase (GAA) with recombinant GlcNAc-phosphotransferase and uncovering enzyme (N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase). SPR analyses using these modified GAAs demonstrate that, unlike the CD-MPR or domain 9 of the CI-MPR, domain 5 exhibits a 14-18-fold higher affinity for Man-P-GlcNAc than Man-6-P, implicating this region of the receptor in targeting phosphodiester-containing lysosomal enzymes to the lysosome.     

Identification of a low affinity mannose 6-phosphate-binding site in domain 5 of the cation-independent mannose 6-phosphate receptor

The 300-kDa cation-independent mannose 6-phosphate receptor (CI-MPR) and the 46-kDa cation-dependent MPR (CD-MPR) are type I integral membrane glycoproteins that play a critical role in the intracellular delivery of newly synthesized mannose 6-phosphate (Man-6-P)-containing acid hydrolases to the lysosome. The extracytoplasmic region of the CI-MPR contains 15 contiguous domains, and the two high affinity ( approximately 1 nm) Man-6-P-binding sites have been mapped to domains 1-3 and 9, with essential residues localized to domains 3 and 9. Domain 5 of the CI-MPR exhibits significant sequence homology to domains 3 and 9 as well as to the CD-MPR. A structure-based sequence alignment was performed that predicts that domain 5 contains the four conserved key residues (Gln, Arg, Glu, and Tyr) identified as essential for carbohydrate recognition by the CD-MPR and domains 3 and 9 of the CI-MPR, but lacks two cysteine residues predicted to form a disulfide bond within the binding pocket. To determine whether domain 5 harbors a carbohydrate-binding site, a construct that encodes domain 5 alone (Dom5His) was expressed in Pichia pastoris. Microarray analysis using 30 different oligosaccharides demonstrated that Dom5His bound specifically to a Man-6-P-containing oligosaccharide (pentamannosyl 6-phosphate). Frontal affinity chromatography showed that the affinity of Dom5His for Man-6-P was approximately 300-fold lower (K(i) = 5.3 mm) than that observed for domains 1-3 and 9. The interaction affinity for the lysosomal enzyme beta-glucuronidase was also much lower (K(d) = 54 microm) as determined by surface plasmon resonance analysis. Taken together, these results demonstrate that the CI-MPR contains a third Man-6-P recognition site that is located in domain 5 and that exhibits lower affinity than the carbohydrate-binding sites present in domains 1-3 and 9.     

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

The cation-dependent mannose 6-phosphate receptor (CD-MPR) is a key component of the lysosomal enzyme targeting system that binds newly synthesized mannose 6-phosphate (Man-6-P)-containing acid hydrolases and transports them to endosomal compartments. The interaction between the MPRs and its ligands is pH-dependent; the homodimeric CD-MPR binds lysosomal enzymes optimally in the pH environment of the trans Golgi network (pH approximately 6.5) and releases its cargo in acidic endosomal compartments (相似文献   

Accumulation of free Neu5Ac-containing complex-type N-glycans in human pancreatic cancers

We have analyzed the structures of glycosphingolipids and intracellular free glycans in human cancers. In our previous study, trace amounts of free N-acetylneuraminic acid (Neu5Ac)-containing complex-type N-glycans with a single GlcNAc at each reducing terminus (Gn1 type) was found to accumulate intracellularly in colorectal cancers, but were undetectable in most normal colorectal epithelial cells. Here, we used cancer glycomic analyses to reveal that substantial amounts of free Neu5Ac-containing complex-type N-glycans, almost all of which were α2,6-Neu5Ac-linked, accumulated in the pancreatic cancer cells from three out of five patients, but were undetectable in normal pancreatic cells from all five cases. These molecular species were mostly composed of five kinds of glycans having a sequence Neu5Ac-Gal-GlcNAc-Man-Man-GlcNAc and one with the following sequence Neu5Ac-Gal-GlcNAc-Man-(Man-)Man-GlcNAc. The most abundant glycan was Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-3Manβ1-4GlcNAc, followed by Neu5Acα2-6Galβ1-4GlcNAcβ1-2Manα1-6Manβ1-4GlcNAc. This is the first study to show unequivocal evidence for the occurrence of free Neu5Ac-linked N-glycans in human cancer tissues. Our findings suggest that free Neu5Ac-linked glycans may serve as a useful tumor marker.     

Strategies for carbohydrate recognition by the mannose 6-phosphate receptors

The two members of the P-type lectin family, the 46 kDa cation-dependent mannose 6-phosphate receptor (CD-MPR) and the 300 kDa cation-independent mannose 6-phosphate receptor (CI-MPR), are ubiquitously expressed throughout the animal kingdom and are distinguished from all other lectins by their ability to recognize phosphorylated mannose residues. The best-characterized function of the MPRs is their ability to direct the delivery of approximately 60 different newly synthesized soluble lysosomal enzymes bearing mannose 6-phosphate (Man-6-P) on their N-linked oligosaccharides to the lysosome. In addition to its intracellular role in lysosome biogenesis, the CI-MPR, but not the CD-MPR, participates in a number of other biological processes by interacting with various molecules at the cell surface. The list of extracellular ligands recognized by this multifunctional receptor has grown to include a diverse spectrum of Man-6-P-containing proteins as well as several non-Man-6-P-containing ligands. Recent structural studies have given us a clearer view of how these two receptors use related, but yet distinct, approaches in the recognition of phosphomannosyl residues.     

Extensive mannose phosphorylation on leukemia inhibitory factor (LIF) controls its extracellular levels by multiple mechanisms

In addition to soluble acid hydrolases, many nonlysosomal proteins have been shown to bear mannose 6-phosphate (Man-6-P) residues. Quantification of the extent of mannose phosphorylation and the relevance to physiological function, however, remain poorly defined. In this study, we investigated the mannose phosphorylation status of leukemia inhibitory factor (LIF), a previously identified high affinity ligand for the cation-independent mannose 6-phosphate receptor (CI-MPR), and we analyzed the effects of this modification on its secretion and uptake in cultured cells. When media from LIF-overexpressing cells were fractionated using a CI-MPR affinity column, 35-45% of the total LIF molecules were bound and specifically eluted with free Man-6-P thus confirming LIF as a bona fide Man-6-P-modified protein. Surprisingly, mass spectrometric analysis of LIF glycopeptides enriched on the CI-MPR column revealed that all six N-glycan sites could be Man-6-P-modified. The relative utilization of these sites, however, was not uniform. Analysis of glycan-deleted LIF mutants demonstrated that loss of glycans bearing the majority of Man-6-P residues leads to higher steady-state levels of secreted LIF. Using mouse embryonic stem cells, we showed that the mannose phosphorylation of LIF mediates its internalization thereby reducing extracellular levels and stimulating embryonic stem cell differentiation. Finally, immunofluorescence experiments indicate that LIF is targeted directly to lysosomes following its biosynthesis, providing another mechanism whereby mannose phosphorylation serves to control extracellular levels of LIF. Failure to modify LIF in the context of mucolipidosis II and its subsequent accumulation in the extracellular space may have important implications for disease pathogenesis.     

Identification of residues essential for carbohydrate recognition by the insulin-like growth factor II/mannose 6-phosphate receptor.

Two distinct mannose 6-phosphate (Man-6-P) receptors (MPRs), the cation-dependent MPR (CD-MPR) and the insulin-like growth factor II/MPR (IGF-II/MPR), recognize a diverse population of Man-6-P-containing ligands. The IGF-II/MPR is a type I transmembrane glycoprotein with a large extracytoplasmic region composed of 15 repeating domains that display sequence identity to each other and to the single extracytoplasmic domain of the CD-MPR. A structure-based sequence alignment of the two distinct Man-6-P-binding sites of the IGF-II/MPR with the CD-MPR implicates several residues of IGF-II/MPR domains 3 and 9 as essential for Man-6-P binding. To test this hypothesis single amino acid substitutions were made in constructs encoding either the N- or the C-terminal Man-6-P-binding sites of the bovine IGF-II/MPR. The mutant IGF-II/MPRs secreted from COS-1 cells were analyzed by pentamannosyl phosphate-agarose affinity chromatography, identifying four residues (Gln-392, Ser-431, Glu-460, and Tyr-465) in domain 3 and four residues (Gln-1292, His-1329, Glu-1354, and Tyr-1360) in domain 9 as essential for Man-6-P recognition. Binding affinity studies using the lysosomal enzyme, beta-glucuronidase, confirmed these results. Together these analyses provide strong evidence that the two Man-6-P-binding sites of the IGF-II/MPR are structurally similar to each other and to the CD-MPR and utilize a similar carbohydrate recognition mechanism.     

Distinctive regulation of the functional linkage between the human cation-independent mannose 6-phosphate receptor and GTP-binding proteins by insulin-like growth factor II and mannose 6-phosphate

The rat insulin-like growth factor II (IGF-II) receptor develops transmembrane signaling functions by directly coupling to a guanine nucleotide-binding protein (G protein) having a 40-kDa alpha subunit, Gi-2, whereas recent studies have indicated that the IGF-II receptor is a molecule identical to the cation-independent mannose 6-phosphate receptor (CI-MPR), a receptor implicated in lysosomal enzyme sorting. In this study, by using vesicles reconstituted with the clonal human CI-MPR and G proteins, we indicated that the CI-MPR could stimulate guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding and GTPase activities of Gi proteins in response to IGF-II. The stimulatory effect of IGF-II on Gi-2 depended on the reconstituted amount of the CI-MPR; it could not be found in vesicles reconstituted with Gi-2 alone; and it was also observed on Gi-1 reconstituted with the CI-MPR in phospholipid vesicles. Of interest, such stimulatory effect was not reproduced by Man-6-P in CI-MPR vesicles reconstituted with either G protein. Furthermore, the affinity for Man-6-P-mediated beta-glucuronidase binding to several kinds of native cell membranes was not reduced by 100 microM GTP gamma S. Instead, however, Man-6-P dose-dependently inhibited IGF-II-induced Gi-2 activation with an IC50 of 6 microM in vesicles reconstituted with the CI-MPR and Gi-2. The action of 100 nM IGF-II was completely abolished by 1 mM Man-6-P. Such an inhibitory effect of Man-6-P was reproduced by 4000 times lower concentrations of beta-glucuronidase or similar concentrations of fructose 1-phosphate, but not by mannose or glucose 6-phosphate. These results indicate that the human CI-MPR has two distinct signaling functions that positively or negatively regulate the activity of Gi-2 in response to the binding of IGF-II or Man-6-P.     

The cation-dependent mannose 6-phosphate receptor. Structural requirements for mannose 6-phosphate binding and oligomerization

The structural requirements for oligomerization and the generation of a functional mannose 6-phosphate (Man-6-P) binding site of the cation-dependent mannose 6-phosphate receptor (CD-MPR) were analyzed. Chemical cross-linking studies on affinity-purified CD-MPR and on solubilized membranes containing the receptor indicate that the CD-MPR exists as a homodimer. To determine whether dimer formation is necessary for the generation of a Man-6-P binding site, a cDNA coding for a truncated receptor consisting of only the signal sequence and the extracytoplasmic domain was constructed and expressed in Xenopus laevis oocytes. The expressed protein was completely soluble, monomeric in structure, and capable of binding phosphomannosyl residues. Like the dimeric native receptor, the truncated receptor can release its ligand at low pH. Ligand blot analysis using bovine testes beta-galactosidase showed that the monomeric form of the CD-MPR from bovine liver and testes is capable of binding Man-6-P. These results indicate that the extracytoplasmic domain of the receptor contains all the information necessary for ligand binding as well as for acid-dependent ligand dissociation and that oligomerization is not required for the formation of a functional Man-6-P binding site. Several different mutant CD-MPRs were generated and expressed in X. laevis oocytes to determine what region of the receptor is involved in oligomerization. Chemical cross-linking analyses of these mutant proteins indicate that the transmembrane domain is important for establishing the quaternary structure of the CD-MPR.     

Oligosaccharides on cathepsin D from porcine spleen

Cathepsin D from porcine spleen contained mannose (3.3%), glucosamine (1.4%), and mannose 6-phosphate (0.08%). Essentially all of the oligosaccharides of cathepsin D could be released by endo-β-N-acetylglucosaminidase H, pointing to oligomajmoside types of structures. Three neutral oligosaccharide fractions, containing 5, 6, and 7 mannose residues, respectively, were isolated by gel permeation chromatography on Bio-Gel P-2. Studies using exoglycosidase digestions and 500-MHz ¹H NMR spectroscopy revealed that their structures are [Manα1 → 2]_{0 or 1}Manα1 → 6[Manα1 → 3]Manα1 → 6[(Manα1 → 2)_{0 or 1}Manα1 → 3]Manβ1 → 4GlcNAcβ1 → 4 GlcNAc. These structures are identical to what have recently been proposed by Takahashi et al. for the major oligosaccharide units of cathepsin D from the same source (T. Takahashi P.G. Schimidt, and J. Tang (1983)J. Biol. Chem.258, 2819–2930), except for the occurrence of two isomeric oligosaccharides containing six mannoses. Only a part (3.4%) of the oligosaccharides were acidic, containing phosphates in monoester linkage. The phosphorylated oligosaccharides also consisted of oligomannoside-type chains which were analogous to, but more heterogeneous in size than the neutral oligosaccharides. Cathepsin D was bound to a mannose- and N-acetylglucosamine-specific lectin (mannan-binding protein) isolated from rabbit liver with the K_i value of 5.4 × 10^?6m.     

Substrate specificity analysis of endoplasmic reticulum glucosidase II using synthetic high mannose-type glycans

Glucosidase II (Glc'ase II) is a glycan-processing enzyme that trims two alpha1,3-linked Glc residues in succession from the glycoprotein oligosaccharide Glc2Man9GlcNAc2 to give Glc1Man9GlcNAc2 and Man9GlcNAc2 in the endoplasmic reticulum (ER). Monoglucosylated glycans, such as Glc1-Man9GlcNAc2, generated by this process play a key role in glycoprotein quality control in the ER, because they are primary ligands for the lectin chaperones calnexin (CNX) and calreticulin (CRT). A precise analysis of the substrate specificity of Glc'ase II is expected to further our understanding of the molecular basis to glycoprotein quality control, because Glc'ase II potentially competes with CNX/CRT for the same glycans, Glc1Man7-9GlcNAc2. In this study, a quantitative analysis of the specificity of Glc'ase II using a series of structurally defined synthetic glycans was carried out. In the presence of CRT, Glc'ase II-mediated trimming from Glc2Man9GlcNAc2 stopped at Glc1Man9GlcNAc2, supporting the notion that the glycan structure delivered to the CNX/CRT cycle is Glc1Man9GlcNAc2. Unexpectedly, our experiments showed that Glc1Man8(B)GlcNAc2 had nearly the same reactivity as Glc1Man9GlcNAc2, which was markedly greater than that of its positional isomer Glc1Man8(C)GlcNAc2. An analysis with glycoprotein-like probes revealed the stepwise formation of Glc1Man9GlcNAc2 and Man9GlcNAc2 from Glc2Man9GlcNAc2, even in the presence of CRT. It was also shown that Glc1Man8(B)GlcNAc2 had even greater reactivity than Glc1Man9GlcNAc2 at the glycoprotein level. Moreover, inhibitory activities by nonglucosylated glycans suggested that Glc'ase II recognized the C arm (Manalpha1, 2Manalpha1, 6Man-) of high mannose-type glycans.     

Complex N-glycans: the story of the “yellow brick road”

The synthesis of complex asparagine-linked glycans (N-glycans) involves a multi-step process that starts with a five mannose N-glycan structure: [Manα1-6(Manα1-3)Manα1-6][Manα1-3]-R where R?=?Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn-protein. N-acetylglucosaminyltransferase I (GlcNAc-TI) first catalyzes addition of GlcNAc in β1-2 linkage to the Manα1-3-R terminus of the five-mannose structure. Mannosidase II then removes two Man residues exposing the Manα1-6 terminus that serves as a substrate for GlcNAc-T II and addition of a second GlcNAcβ1-2 residue. The resulting structure is the complex N-glycan: GlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3)-R. This structure is the precursor to a large assortment of branched complex N-glycans involving four more N-acetylglucosaminyltransferases. This short review describes the experiments (done in the early 1970s) that led to the discovery of GlcNAc-TI and II.     

Mechanisms for high affinity mannose 6-phosphate ligand binding to the insulin-like growth factor II/mannose 6-phosphate receptor

The two mannose 6-phosphate (Man-6-P) binding domains of the insulin-like growth factor II/mannose 6-phosphate receptor (Man-6-P/IGF2R), located in extracytoplasmic repeats 1-3 and 7-9, are capable of binding Man-6-P with low affinity and glycoproteins that contain more than one Man-6-P residue with high affinity. High affinity multivalent ligand binding sites could be formed through two possible mechanisms: the interaction of two Man-6-P binding domains within one Man-6-P/IGF2R molecule or by receptor oligomerization. To discriminate between these mechanisms, truncated FLAG epitope-tagged Man-6-P/IGF2R constructs, containing one or both of the Man-6-P binding domains, were expressed in 293T cells, and characterized for binding of pentamannose phosphate-bovine serum albumin (PMP-BSA), a pseudoglycoprotein bearing multiple Man-6-P residues. A construct containing all 15 repeats of the Man-6-P/IGF2R extracytoplasmic domain bound PMP-BSA with the same affinity as the full-length receptor (K(d) = 0.54 nm) with a curvilinear Scatchard plot. The presence of excess unlabeled PMP-BSA increased the dissociation rate of pre-formed (125)I-PMP-BSA/receptor complexes, suggesting negative cooperativity in multivalent ligand binding and affirming the role of multiple Man-6-P/IGF2R binding domains in forming high affinity binding sites. Truncated receptors containing only one Man-6-P binding domain and mutant receptor constructs, containing an Arg(1325) --> Ala mutation that eliminates binding to the repeats 7-9 binding domain, formed high affinity PMP-BSA binding, but with reduced stoichiometries. Collectively, these observations suggest that alignment of Man-6-P binding domains of separate Man-6-P/IGF2R molecules is responsible for the formation of high affinity Man-6-P binding sites and provide functional evidence for Man-6-P/IGF2R oligomerization.     

Comparative studies of asparagine-linked oligosaccharide structures of rat liver microsomal and lysosomal beta-glucuronidases

The sugar chains of microsomal and lysosomal β-glucuronidases of rat liver were studied by endo-β-N-acetylglucosaminidase H digestion and by hydrazinolysis. Only a part of the oligosaccharides released from microsomal β-glucuronidase was an acidic component. The acidic component was not hydrolyzed by sialidase and by calf intestinal and Escherichia coli alkaline phosphatases, but was converted to a neutral component by phosphatase digestion after mild acid treatment indicating the presence of a phosphodiester group. The neutral oligosaccharide portion of microsomal enzyme was a mixture of five high mannose-type sugar chains: (Manα1 → 2)_0~4 [Manα1 → 6(Manα1 → 3)Manα1 → 6(Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4GlcNAc]. In contrast, lysosomal enzyme contains only Manα1 → 6 (Manα1 → 3) Manα1 → 6(Manα1 → 3) Manβ1 → 4GlcNAcβ1 → 4GlcNAc. The result indicates that removal of α1 → 2-linked mannosyl residues from (Manα1 → 2)₄[Manα1 → 6(Manα1 → 3)Manα1 → 6(Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4GlcNAc → Asn] starts already in the endoplasmic reticulum of rat liver.     

Distribution of newly synthesized lysosomal enzymes in the endocytic pathway of normal rat kidney cells

We have investigated the distribution of newly synthesized lysosomal enzymes in endocytic compartments of normal rat kidney (NRK) cells. The mannose-6-phosphate (Man6-P) containing lysosomal enzymes could be iodinated in situ after internalization of lactoperoxidase (LPO) by fluid phase endocytosis and isolated on CI-MPR affinity columns. For EM studies, the ectodomain of the CI-MPR conjugated to colloidal gold was used as a probe specific for the phosphomannosyl marker of the newly synthesized hydrolases. In NRK cells, approximately 20-40% of the phosphorylated hydrolases present in the entire pathway were found in early endocytic structures proximal to the 18 degrees C temperature block including early endosomes. These structures were characterized by a low content of endogenous CI-MPR and were accessible to fluid phase markers internalized for 5-15 min at 37 degrees C. The bulk of the phosphorylated lysosomal enzymes was found in late endocytic structures distal to the 18 degrees C block, rich in endogenous CI-MPR and accessible to endocytic markers internalized for 30-60 min at 37 degrees C. The CI-MPR negative lysosomes were devoid of phosphorylated hydrolases. This distribution was unchanged in cells treated with Man6-P to block recapture of secreted lysosomal enzymes. However, lysosomal enzymes were no longer detected in the early endosomal elements of cells treated with cycloheximide. Immunoprecipitation of cathepsin D from early endosomes of pulse-labeled cells showed that this hydrolase is a transient component of this compartment. These data indicate that in NRK cells, the earliest point of convergence of the lysosomal biosynthetic and the endocytic pathways is the early endosome.     

Specificity towards oligomannoside and hybrid type glycans of the endo-β-N-acetylglucosaminidase B from the basidiomy ceteSporotrichum dimorphosporum

We have previously shown that an endo--N-acetylglucosaminidase (EC 3.2.1.96) named Endo B, isolated from culture filtrates of the basidiomyceteSporotrichum dimorphosporum cleaves asialo-, and to some extent, monosialylated bi-antennary glycans of theN-acetyllactosamine type linked to the asparagine residue of peptide or protein moieties [Bouquelet S, Strecker G, Montreuil J, Spik G (1980) Biochimie 62:43–49]. In the present paper, the substrate specificity of the enzyme towards oligomannoside and hybrid type glycans has been analyzed. The results obtained indicate that ovalbumin glycopeptides containing four to seven mannose residues and bovine lactotransferrin glycopeptides containing four to nine mannose residues were completely hydrolyzed by the enzyme. The degree of cleavage was variable among hybrid type structures, since glycopeptides containing the following glycans: (Gal)₁(GlcNAc)₃(Man)₅(GlcNAc)₂; (GlcNAc)₃(Man)₅(GlcNAc)₂; (GlcNAc)₃(Man)₄(GlcNAc)₂ were not hydrolyzed by the enzyme while the percentage of hydrolysis of a glycopeptide containing (GlcNAc)₂(Man)₅(GlcNAc)₂ glycan reached 90%. The bovine lactotransferrin was partially deglycosylated (40%) in the absence of non-ionic detergent while native ovalbumin glycoprotein was not hydrolyzed by the enzyme.The oligomannoside-and theN-acetyllactosamine-type degrading activities present in the culture filtrates were not separated at any step of the purification procedure. Both activities were eluted as a single component with an apparent molecular mass of 89 kDa suggesting that they are located on the same enzyme molecule.Endo B represents a powerful tool for removing oligomannoside-andN-acetyllactosamine-type glycans fromN-glycopeptides andN-glycoproteins. Moreover, advantages in the use of Endo B in a soluble form as well as in an immobilized form result in its high activity and in its stability to heat denaturation and storage.Abbreviations Gal d-galactose - Man d-mannose - GlcNAc N-acetyl-d-glucosamine - Con A concanavalin A - Asn asparagine - GLC gas liquid chromatography - TLC thin layer chromatography - Endo endo--N-acetylglucosaminidase - Endo B endo--N-acetylglucosaminidase isolated fromSporotrichum dimorphosporum - PBE polybuffer exchanger - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Binding receptors for α-l-fucosidase in human B-lymphoid cell lines

An established mechanism for directing newly made acid hydrolases to lysosomes involves acquisition of mannose 6-phosphate residues by the carbohydrate portion of acid hydrolases followed by binding to specific membrane-bound transport receptors and delivery to lysosomes. Two distinct phosphomannosyl receptors (CI-MPR and CD-MPR) have been identified. Alternative mechanisms for trafficking acid hydrolases exist. This report examines means for the possible receptor-mediated intracellular transport of -l-fucosidase in lymphoid cells. The binding of -l-fucosidase to intact cells and to total cell membrane preparations, in conjunction with immunoassays of solubilized membrane preparations, revealed the presence of CI-MPR and CD-MPR on human lymphoid and fibroblast cell lines. The mean level of CD-MPR in nine lymphoid cell lines was 7.2-fold greater than CI-MPR. The mean level of CI-MPR in two fibroblast lines was 3.8-fold greater than CD-MPR. The mean content of CI-MPR was 19.5-fold greater in the fibroblasts than in the lymphoid cells. The CD-MPR content of fibroblasts and lymphoid cells was nearly equivalent. Among these cell lines were a fibroblast and a lymphoid line from the same individual. These results indicate that human B-lymphoid cells are deficient in CI-MPR and suggest that modulation of expression of CI-MPR and CD-MPR in lymphoid cells differs from that in fibroblasts, including cell lines with identical genomes. No specific receptor capable of binding -l-fucosidase independent of mannose 6-phosphate was demonstrable, despite published results that support the existence of a mannose 6-phosphate independent trafficking mechanism in lymphoid cells for this enzyme.     

Structural analysis of trisialylated biantennary glycans isolated from mouse serum transferrin: Characterization of the sequence Neu5Gc(α2-3)Gal(β1-3)[Neu5Gc(α2-6)]GlcNAc(β1-2)Man

Five variants of mouse serum transferrin (mTf, designated mTf-I to mTf-V) with respect to carbohydrate composition have been isolated by DEAE-cellulose chromatography in the following relative percentages: mTf-I: 0.55; mTf-II: 0.79; mTf-III: 71.80; mTf-VI: 21.90 and mTf-V: 4.96. The primary structures of the major glycans from mTf-III and mTf-IV were determined by methylation analysis and ¹H-nuclear magnetic resonance (NMR) spectroscopy. All glycans possessed a common trimannosyl-N,N′-diacetylchitobiose core. From the glycovariant mTf-III two isomers of a conventional biantennary N-acetyllactosamine type were isolated, in which two N-glycolylneuraminic acid (Neu5Gc) residues are linked to galactose either by a (α2-6) or (α2-3) linkage. A subpopulation of this glycovariant contains a fucose residue (α1-6)-linked to GlcNAc-1. The structure of the major glycan found in variant mTf-IV contained an additional Neu5Gc and possessed the following new type of linkage: Neu5Gc(α2-3)Gal(β1-3)[Neu5Gc(α2-6)]GlcNAc(β1-2)Man(α1-3). In addition to this glycan, a minor compound contained the same antennae linked to Man(α1-6). In fraction mTf-V, which was found to be very heterogeneous by ¹H NMR analysis, carbohydrate composition and methylation analysis suggested the presence of tri′-antennary glycans sialylated by Neu5Gc α-2,6- and α-2,3-linked to the terminal galactose residues. In summary, mTf glycans differed from those of other analyzed mammalian transferrins by the presence of Neu5Gc and by a Neu5Gc(α2-6)GlcNAc linkage in trisialylated biantennary structures, reflecting in mouse liver, a high activity of CMP-Neu5Ac hydroxylase and (α2-6)GlcNAc sialyltransferase.     

The interaction of phosphorylated oligosaccharides and lysosomal enzymes with bovine liver cation-dependent mannose 6-phosphate receptor

We have analyzed the interaction of phosphorylated oligosaccharides and lysosomal enzymes with immobilized bovine liver cation-dependent mannose-6-P receptor. Oligosaccharides with phosphomonoesters were the only species that interacted with the receptor, and molecules with two phosphomonoesters showed the best binding. Lysosomal enzymes with several oligosaccharides containing only one phosphomonoester had a higher affinity for the receptor than did the isolated oligosaccharides, indicating the possible importance of multivalent interactions between weakly binding ligands and the receptor. The binding of a mixture of phosphorylated lysosomal enzymes to the cation-dependent Man-6-P receptor was markedly influenced by pH. At pH 6.3, almost all of the lysosomal enzymes bound to the receptor; whereas at pH 7.0-7.5, approximately one-third of the material passed through the column, one-third interacted weakly, and one-third bound tightly. The distribution of individual lysosomal enzyme activities was similar to that of the total material. The species of phosphorylated oligosaccharides present on the lysosomal enzymes which interacted poorly with the receptor were similar to those found on the tightly bound material and included species of oligosaccharides with two phosphomonoester groups. Isolated oligosaccharides of this type bound to the receptor over the entire pH range tested. These findings indicate that at neutral pH the phosphorylated oligosaccharides on some lysosomal enzyme molecules are oriented in a manner which makes them inaccessible to the binding site of the cation-dependent Man-6-P receptor. Since the same enzymes bind to the cation-independent Man-6-P receptor at neutral pH, at least a portion of the phosphomannosyl residues must be exposed. We conclude that small variations in the pH of the Golgi compartment where lysosomal enzymes bind to the receptors could potentially modulate the extent of binding to the two receptors.