Phosphomannomutase activity in congenital disorders of glycosylation type Ia determined by direct analysis of the interconversion of mannose-1-phosphate to mannose-6-phosphate by high-pH anion-exchange chromatography with pulsed amperometric detection

Congenital disorders of glycosylation (CDG) are a group of multisystemic disorders resulting from defects in the synthesis and processing of N-linked oligosaccharides. The most common form, CDG type Ia (CDG-Ia), results from a deficiency of the enzyme phosphomannomutase (PMM). PMM converts mannose 6-phosphate (man-6-P) to mannose-1-phosphate (man-1-P), which is required for the synthesis of GDP-mannose, a substrate for dolichol-linked oligosaccharide synthesis. The traditional assay for PMM, a coupled enzyme system based on the reduction of NADP(+) to NADPH using man-1-P as a substrate, has limitations in accuracy and reproducibility. Therefore, a more sensitive, direct test for PMM activity, based on the detection of the conversion of man-1-P to man-6-P by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), was developed. Using this assay, the activity of PMM was markedly deficient in fibroblasts and lymphoblasts from 23 patients with CDG-Ia (range 0-15.3% of control, average 4.9+/-4.7%) and also decreased in seven obligate heterozygotes (range 33.0-72.0% of control, average 52.2+/-14.7%). Unlike the spectrophotometric method, there was no overlap in PMM activity among patients, obligate heterozygotes, or controls. Thus, the PMM assay based on HPAEC-PAD has increased utility in the clinical setting, and can be used, together with transferrin isoelectric focusing, to diagnose patients with CDG-Ia and to identify heterozygotes when clinically indicated.     

A fluorescence assay for alpha-1,2-mannosidases involved in glycoprotein processing reactions

A highly specific, sensitive, and convenient fluorescence assay for alpha-1,2-mannosidases involved in glycoprotein processing reactions is described. The assay utilizes a coupled enzyme system to determine the amount of free mannose liberated from the disaccharide O-methyl-2-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside by the alpha-1,2-mannosidase. The assay was used to determine the substrate specificity of a calcium ion-activated alpha-1,2-mannosidase purified from rabbit liver microsomes. The microsomal mannosidase was specific for hydrolysis of the alpha-1,2 linkage. The mannosyl linkages in alpha-1,3- and alpha-1,6-linked methyl-disaccharides, in methyl-alpha-D-mannopyranoside, and in yeast mannan were hydrolyzed at rates of 2% or less than that noted with the alpha-1,2-linked disaccharide. Mannosidase activity was linear with time and was proportional to enzyme concentration. The Km for the alpha-1,2-linked methyl-disaccharide is 0.5 mM.     

A Quantitative Mannose 6-Phosphate Receptor-Basedin VitroAssay for Recombinant HumanN-Acetylgalactosamine-4-sulfatase

An assay was developed, using two similar formats, to simultaneously measure both the lysosomal targeting receptor binding and enzyme activity of the recombinant human enzymeN-acetylgalactosamine-4-sulfatase. This assay also has potential application for all phosphorylated lysosomal enzymes that contain mannose-6-phosphate residues. The receptor was either purified from fetal bovine sera then adsorbed, or producedin situby growing and fixing diploid human fibroblast-like cells, to a solid phase. The enzyme substrate was 4-methylumbelliferyl sulfate which fluoresces after cleavage of the sulfate moiety. Both the precursor and mature forms of the recombinant enzyme were used to demonstrate the specificity and usefulness of the assay. The assay is rapid and sensitive and has a wide dynamic range. Association between the receptor and the mannose-6-phosphate residues was abrogated in the presence of a competitive inhibitor, mannose 6-phosphate. However, partial activity was still measured when the mature enzyme was incubated in the presence of mannose 6-phosphate when using the fixed fibroblast format. This would indicate that the recombinant enzymes contain at least one terminal sugar moiety other than mannose 6-phosphate which can recognize receptors on the surface of human fibroblast-like cells. Other possible applications of this assay are also discussed.     

Cellular repressor of E1A-stimulated genes is a bona fide lysosomal protein which undergoes proteolytic maturation during its biosynthesis

Cellular repressor of E1A-stimulated genes (CREG) has been reported to be a secretory glycoprotein implicated in cellular growth and differentiation. We now show that CREG is predominantly localized within intracellular compartments. Intracellular CREG was found to lack an N-terminal peptide present in the secreted form of the protein. In contrast to normal cells, CREG is largely secreted by fibroblasts missing both mannose 6-phosphate receptors. This is not observed in cells lacking only one of them. Mass spectrometric analysis of recombinant CREG revealed that the protein contains phosphorylated oligosaccharides at either of its two N-glycosylation sites. Cellular CREG was found to cosediment with lysosomal markers upon subcellular fractionation by density-gradient centrifugation. In fibroblasts expressing a CREG-GFP fusion construct, the heterologous protein was detected in compartments containing lysosomal proteins. Immunolocalization of endogenous CREG confirmed that intracellular CREG is localized in lysosomes. Proteolytic processing of intracellular CREG involves the action of lysosomal cysteine proteinases. These results establish that CREG is a lysosomal protein that undergoes proteolytic maturation in the course of its biosynthesis, carries the mannose 6-phosphate recognition marker and depends on the interaction with mannose 6-phosphate receptors for efficient delivery to lysosomes.     

A novel fluorometric assay for ADP-ribose pyrophosphatase activity

ADP-ribose pyrophosphatase (ADPRase) hydrolyzes ADP-ribose to ribose-5-phosphate and AMP. The ADPRase activity have been assessed by coupling the reaction to alkaline phosphatase and colorimetrically measuring the amount of inorganic phosphate released from AMP that is one of the products of ADPRase. Another but less sensitive colorimetric method has been employed: the reaction mixture was treated with charcoal to adsorb the adenine-containing compounds such as AMP and ADPR and subsequently remaining ribose-5-phosphate was measured colorimetrically. However, the measurement of inorganic phosphate cannot be feasible to assay ADPRase in phosphate-containing samples and the determination of ribose-5-phosphate also is less sensitive. Here we develop a fluorescent assay for ADPRase that utilizes 1, N(6)-etheno ADP-ribose, a fluorescent analogue of ADP-ribose. This method measures fluorescent 1, N(6)-etheno adenosine that is produced by coupling the hydrolysis of 1, N(6)-etheno ADP-ribose to dephosphorylation with alkaline phosphatase. The fluorometric assay is comparable in sensitivity and useful for ADPRase assay in phosphate-containing samples.     

Identification of free mannose and partial characterization of a mannose-6-phosphate isomerase from Lilium longiflorum bulbs

The existence of free mannose in storage bulbs of Lilium longiflorum Thunb, was established using preparative high performance liquid chromatography, gas chromatography and gas chromatography-mass spectroscopy. Free mannose was not detected in developing (importing) bulb tissues. Mannose, a relatively rare hexose in plant tissue, probably arises from the hydrolysis of glucomannan, a hemicellulosic carbohydrate polymer known to be present in Lilium storage tissues. A calculation of total mannose residues per bulb (prior to versus after reserve hydrolysis and export) indicated that mannose is metabolized, probably in sucrose biosynthesis. A mannose-6-phosphate isomerase (EC 5.3.1.8) was isolated from Lilium bulbs and purified 155-fold with 29% yield. The molecular weight of the enzyme was estimated by gel filtration to be 64 kDa, and the K_m for mannose-6-phosphate was 0.42 m M . It is concluded that glucomannan is functioning as a reserve carbohydrate in Lilium storage tissues and that the mannose-6-phosphate isomerase is responsible for the entry of mannose into the sucrose biosynthetic pathway.     

Natural killer cell-mediated cytotoxicity does not depend on recognition of mannose 6-phosphate residues

Interaction of mannose 6-phosphate-specific receptors with their ligands has been suggested to be essential for natural killer cell (NK)-mediated cytotoxicity. Indeed, mannose 6-phosphate-specific receptors and ligands bearing mannose 6-phosphate residues are demonstrable on human peripheral blood leukocytes with NK activity as well as on K-562 NK target cells, allowing at least in principle such an interaction. It can also be shown that NK activity of human peripheral blood leukocytes is inhibited by mannose 6-phosphate. The following observations, however, exclude an essential role of the mannose 6-phosphate receptor-ligand system in NK cell-mediated cytotoxicity. 1) NK cytotoxicity is sensitive to a broad range of structurally unrelated sugar phosphates. 2) NK activity is normal in patients with I cell disease (mucolipidosis II), which due to a genetic defect are unable to synthesize the ligands for the mannose 6-phosphate-specific receptor. 3) NK cytotoxicity is not inhibited by an antiserum against the mannose 6-phosphate receptor, which blocks the receptor function.     

Proliferin secreted by cultured cells binds to mannose 6-phosphate receptors

Proliferin is a prolactin-related glycoprotein secreted by proliferating mouse cell lines and by mouse placenta. In an attempt to identify target sites for proliferin action, we looked for proliferin receptors in murine fetal and maternal tissues during pregnancy using proliferin purified from the conditioned medium of a constructed Chinese hamster ovary cell line carrying amplified copies of proliferin cDNA. Purified proliferin bound to membrane preparations from fetal or maternal liver and from placenta with a Kd of 1 to 2 nM. The amount of proliferin bound per microgram of membrane protein varied markedly during pregnancy; maximal binding to day 16 fetal liver membranes was approximately 25 times that to liver membranes from adult animals. Binding to fetal and maternal receptors was specifically and completely inhibited by mannose 6-phosphate, with half-maximal inhibition at 10 microM. Furthermore, non-glycosylated proliferin did not inhibit the binding of the glycosylated protein. A approximately 300 Kd proliferin receptor was purified from the liver of pregnant mice using a proliferin affinity column and elution with mannose 6-phosphate. This receptor reacted with antibodies directed against the rat cation-independent mannose 6-phosphate receptor. We conclude that 1) proliferin secreted by cultured cell binds to cation-independent mannose 6-phosphate receptors and therefore may be a lysosomal protein or targeted to lysosomes, and 2) the concentration or activity of mannose 6-phosphate receptors in murine fetal and maternal liver and in placenta is regulated during pregnancy.     

The 52-kDa estrogen-induced protein secreted by MCF7 cells is a lysosomal acidic protease

An estrogen-induced 52-kDa glycoprotein secreted by human breast cancer cells and able to autostimulate the growth of MCF7 cells has been purified, using monoclonal antibodies, and characterized. The protein contains mannose 6-phosphate signals on its N-linked high-mannose chains, suggesting that it is a lysosomal enzyme. Both the secreted 52-kDa protein and its processed cellular forms (52-, 48- and 34-kDa) were identified as carboxyl proteinases having an optimal activity at pH 3.5 and being specifically inhibited by pepstatin. This protease is characterized by its inducibility by estrogens and its high concentration in proliferative benign and malignant mammary tissue, when detected by immunohistochemistry. The estrogen-induced secretion of this protease may help to understand how estrogens stimulate mammary tumor growth and/or invasion.     

Possible mechanism of mannose inhibition of sucrose-supported growth in N2-fixing Azotobacter vinelandii

When mannose was added to a sucrose-supported culture of Azotobacter vinelandii under N2-fixing conditions, cell growth was inhibited. The degree of inhibition was proportional to the amount of mannose and to the aeration rate (T.-Y. Wong, Appl. Environ. Microbiol. 54:473-475, 1988). In this report, we demonstrate that once inside the cell, mannose was phosphorylated to mannose 6-phosphate. It was then isomerized to fructose 6-phosphate and to glucose 6-phosphate. Mannose inhibited sucrose uptake noncompetitively. The decrease in sucrose uptake after mannose addition coincided with a lower rate of respiration and a decrease in nitrogenase activity. The decrease in sucrose uptake and in the ATP pool may decrease the electron flow and reduce protection of the nitrogenase from O2. Cells became very sensitive to O2, and therefore, cell growth was inhibited under high aeration conditions.     

Possible mechanism of mannose inhibition of sucrose-supported growth in N2-fixing Azotobacter vinelandii.

When mannose was added to a sucrose-supported culture of Azotobacter vinelandii under N2-fixing conditions, cell growth was inhibited. The degree of inhibition was proportional to the amount of mannose and to the aeration rate (T.-Y. Wong, Appl. Environ. Microbiol. 54:473-475, 1988). In this report, we demonstrate that once inside the cell, mannose was phosphorylated to mannose 6-phosphate. It was then isomerized to fructose 6-phosphate and to glucose 6-phosphate. Mannose inhibited sucrose uptake noncompetitively. The decrease in sucrose uptake after mannose addition coincided with a lower rate of respiration and a decrease in nitrogenase activity. The decrease in sucrose uptake and in the ATP pool may decrease the electron flow and reduce protection of the nitrogenase from O2. Cells became very sensitive to O2, and therefore, cell growth was inhibited under high aeration conditions.     

Further Studies of Pollen Wall Glycoproteins in Cucurbita pepo L.

Samples of pollen wall protein of Cucurbita pepo were prepared as reported in previous paper. Gas chromoatographic analyses snowed that the carbohydrate fraction of the pollen wall glycoprotcin contained 20.4% rhamnose, 15.3% fucose, 11% mannose, 11% galactose, 31% glucose, 4% arabinose and traces of xylose. The glycoproteins were further purified by Con. A affinity chromatography, Isoelectric focussing electrophoresis of the purified sample showed 3 PAS-positive bands, with respective PI 5.2, 6.0 and 6.3. The glycoprotein samples were subjected to hydrolysis with 6N HC1. After hydrolysis, the mixture was analyzed for amino acid composition with Backman 121-MB automatic amino acid analyzer, Results show the amino acid composition of the 3 glycoprotein was very similar, They all have glycine, glutamic acid and serine as their major component (these three amino acids constitute 50–60% of the total amino acids); and they all contain very small amount of methionine, phenylalanine, isoleucine and tyrosine. The lysine content of each glycoprotein is consistent with its respective PI, the glycoprotein which contains more lysine has higher PI.     

Quantitation of Mr 46000 and Mr 300000 mannose 6-phosphate receptors in human cells and tissues.

A highly specific enzyme-linked immunosorbent assay for quantitation of the Mr 46000 Da and Mr 300000 Da mannose 6-phosphate receptors was developed. The assay allows to detect ng amounts of human mannose 6-phosphate receptors. Analysis of human cells and tissues revealed significant differences in their contents of the two mannose 6-phosphate receptors, normalized for total cell protein. The ratio of the two mannose 6-phosphate receptors also differed among cells and tissues, suggesting that their steady state concentrations are regulated independently.     

Presence of mannose phosphate on the epidermal growth factor receptor in A-431 cells

In this report, we demonstrate a novel post-translational modification of the epidermal growth factor (EGF) receptor. This modification involves the presence of phosphate, previously thought to exist only on amino acid residues in the EGF receptor, on oligosaccharides of the receptor. We have utilized several independent approaches to determine that mannose phosphate is present on the EGF receptor in A-431 cells. Following metabolic labeling with 32P, immunoisolation of the EGF receptor, and digestion with Pronase radioactivity was determined to be present on high mannose type oligosaccharides by concanavalin A chromatography. Also, after acid hydrolysis of in vivo 32P-labeled EGF receptor, radioactivity was detected that co-migrated with mannose 6-phosphate on two-dimensional thin layer electrophoresis. This radiolabeled material co-eluted with a mannose 6-phosphate standard from a high pressure liquid chromatography anion exchange column. Last, an acid hydrolysate of [3H]mannose-labeled EGF receptor contained two radiolabeled fractions, as analyzed by thin layer electrophoresis, and the radioactivity in one of these fractions was substantially reduced by alkaline phosphatase treatment prior to electrophoresis. These experiments indicate that the mature EGF receptor in A-431 cells contains mannose phosphate. This is a novel modification for membrane receptors and has only been reported previously for lysosomal enzymes and a few secreted proteins.     

A novel mass spectrometric procedure for the rapid determination of the types of carbohydrate chains present in glycoproteins: application to alpha-galactosidase I from Vicia faba seeds

A fast atom bombardment mass spectrometric protocol has been developed to determine the type of oligosaccharide chain present in glycoproteins. The procedure is based on acetolysis of the intact glycoconjugate, extraction of the peracetylated carbohydrate fragments and analysis by fast atom bombardment mass spectrometry. The molecular ions present in the FAB spectra uniquely define the composition of the oligosaccharides with respect to hexose, aminohexose and sialic acid content. High mannose oligosaccharides yield a series of peracetylated hexose oligomers whereas complex-type oligosaccharides afford a series of N-acetyl-lactosamine containing species. Fucosylation is usually not detected but sialylated oligosaccharides are readily identified and the type of sialic acid is also defined. The method has been tested on three glycoproteins of known structure - fetuin, ribonuclease B and erythrocyte Band 3 - and on a glycoprotein of unknown structure - alpha-galactosidase I, an enzyme lectin from Vicia faba. The latter is shown to contain high mannose carbohydrate chains.     

Recognition of Dictyostelium discoideum lysosomal enzymes is conferred by the amino-terminal carbohydrate binding site of the insulin-like growth factor II/mannose 6-phosphate receptor

The insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/MPR) is a type I glycoprotein that mediates both the intracellular sorting of lysosomal enzymes bearing mannose 6-phosphate (Man-6-P) residues to the lysosome and the bioavailability of IGF-II. The extracytoplasmic region of the IGF-II/MPR contains 15 repeating domains; the two carbohydrate recognition domains (CRDs) have been localized to domains 1-3 and 7-9, and the high-affinity IGF-II binding site maps to domain 11. To characterize the carbohydrate binding properties of the IGF-II/MPR, regions of the receptor encompassing the individual CRDs were produced in a baculovirus expression system. Characterization of the recombinant proteins revealed that the pH optimum for carbohydrate binding is significantly more acidic for the carboxyl-terminal CRD than for the amino-terminal CRD (i.e., pH 6.4-6.5 vs 6.9). Equilibrium binding studies demonstrated that the two CRDs exhibit a similar affinity for Man-6-P. Furthermore, substitution of the conserved arginine residue in domain 3 (R435) or in domain 9 (R1334) with alanine resulted in a similar >1000-fold decrease in the affinity for the lysosomal enzyme, beta-glucuronidase. In contrast, the two CRDs differ dramatically in their ability to recognize the distinctive modifications (i.e., mannose 6-sulfate and Man-6-P methyl ester) found on Dictyostelium discoideum lysosomal enzymes: the amino-terminal CRD binds mannose 6-sulfate and Man-6-P methyl ester with a 14-55-fold higher affinity than the carboxyl-terminal CRD. Taken together, these results demonstrate that the IGF-II/MPR contains two functionally distinct CRDs.     

Intracellular hexose-6-phosphate:phosphohydrolase from Streptococcus lactis: purification, properties, and function.

An intracellular hexose 6-phosphate:phosphohydrolase (EC 3.1.3.2) has been purified from Streptococcus lactis K1. Polyacrylamide disc gel electrophoresis of the purified enzyme revealed one major activity staining protein and one minor inactive band. The Mr determined by gel permeation chromatography was 36,500, but sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single polypeptide of apparent Mr 60,000. The enzyme exhibited a marked preference for hexose 6-phosphates, and the rate of substrate hydrolysis (at 5 mM concentration) decreased in the order, galactose 6-phosphate greater than 2-deoxy-D-glucose 6-phosphate greater than fructose 6-phosphate greater than mannose 6-phosphate greater than glucose 6-phosphate. Hexose 1-phosphates, p-nitrophenylphosphate, pyrophosphate, and nucleotides were not hydrolyzed at a significant rate. In addition, the glycolytic intermediates comprising the intracellular phosphoenolpyruvate potential in the starved cells (phosphoenolpyruvate and 2- and 3-phosphoglyceric acids) were not substrates for the phosphatase. Throughout the isolation, the hexose 6-phosphate:phosphohydrolase was stabilized by Mn2+ ion, and the purified enzyme was dependent upon Mn2+, Mg2+, Fe2+, or Co2+ for activation. Other divalent metal ions including Pb2+, Cu2+, Zn2+, Cd2+, Ca2+, Ba2+, Sr2+, and Ni2+ were unable to activate the enzyme, and the first four cations were potent inhibitors. Enzymatic hydrolysis of 2-deoxy-D-glucose 6-phosphate was inhibited by fluoride when Mg2+ was included in the assay, but only slight inhibition occurred in the presence of Mn2+, Fe2+, or Co2+. The inhibitory effect of Mg2+ plus fluoride was specifically and completely reversed by Fe2+ ion. The hexose 6-phosphate:phosphohydrolase catalyzes the in vivo hydrolysis of 2-deoxy-D-glucose 6-phosphate in stage II of the phosphoenolpyruvate-dependent futile cycle in S. lactis (J. Thompson and B. M. Chassy, J. Bacteriol. 151:1454-1465, 1982).     

31P-NMR and 13C-NMR studies of mannose metabolism in Plesiomonas shigelloides. Toxic effect of mannose on growth.

The metabolism of mannose was examined in resting cells in vivo using 13C-NMR and 31P-NMR spectroscopy, in cell-free extracts in vitro using 31P-NMR spectroscopy, and by enzyme assays. Plesiomonas shigelloides was shown to transport mannose by a phosphoenolpyruvate-dependent phosphotransferase system producing mannose 6-phosphate. However, a toxic effect was observed when P. shigelloides was grown in the presence of mannose. Investigation of mannose metabolism using in vivo 13C NMR showed mannose 6-phosphate accumulation without further metabolism. In contrast, glucose was quickly metabolized under the same conditions to lactate, ethanol, acetate and succinate. Extracts of P. shigelloides exhibited no mannose-6-phosphate isomerase activity whereas the key enzyme of the Embden-Meyerhof pathway (6-phosphofructokinase) was found. This result explains the mannose 6-phosphate accumulation observed in cells grown on mannose. The levels of phosphoenolpyruvate and Pi were estimated by in vivo 31P-NMR spectroscopy. The intracellular concentrations of phosphoenolpyruvate and Pi were relatively constant in both starved cells and mannose-metabolizing cells. In glucose-metabolizing cells, the phosphoenolpyruvate concentration was lower, and about 80% of the Pi was used during the first 10 min. It thus appears that the toxic effect of mannose on growth is not due to energy depletion but probably to a toxic effect of mannose 6-phosphate.     

Characterization of a structural glycoprotein from bovine ligamentum nuchae exhibiting dual amine oxidase activity

A structural glycoprotein has been extracted from bovine ligamentum nuchae by using 5 M guanidine hydrochloride containing a disulfide bond reducing agent and purified by preparative gel electrophoresis. The isolated material appeared to be monodisperse, with a molecular weight of approximately 34000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by analytical ultracentrifugation. It contains 10% carbohydrate comprising mannose, N-acetylglucosamine, galactose, and sialic acid in a 6:5:3:3 molar ratio. The glycoprotein has been assayed for peptidyl-lysine oxidase activity by using [3H]lysine-aortic elastin, prepared from 15- to 17-day-old chick embryos, as a substrate. In the absence of free lysine, the specific activity of the preparation over a 2-h incubation was approximately 60 X 10(4) dpm/mg of purified protein. Addition of 10 mM lysine resulted in an approximately 50% decrease in the specific activity. Free lysine was shown to act as a substrate for the glycoprotein preparation as indicated by control experiments using [3H]lysine in place of the aortic substrate. These results demonstrate that the glycoprotein exhibits a dual amine oxidase activity. In the presence of 0.27 mM beta-aminopropionitrile fumarate, a concentration which completely inhibits peptidyl-lysine oxidase activity in other lysyl oxidases, the glycoprotein preparation was inhibited by approximately 14%. In the absence of 5 M guanidine hydrochloride and reducing agent, the glycoprotein undergoes aggregation which in the presences of copper ions results in the formation of cylindrical tactoids, the diameter of which (11 nm) corresponds closely to that of the fibrils which in the majority of connective tissue matrices constitute the microfibrillar component mainly associated with elastic fibers.     

Ligand interactions of the cation-independent mannose 6-phosphate receptor. The stoichiometry of mannose 6-phosphate binding

The interaction of the bovine cation-independent mannose 6-phosphate receptor with a variety of phosphorylated ligands has been studied using equilibrium dialysis and immobilized receptor to measure ligand binding. The dissociation constants for mannose 6-phosphate, pentamannose phosphate, bovine testes beta-galactosidase, and a high mannose oligosaccharide with two phosphomonoesters were 7 X 10(-6) M, 6 X 10(-6) M, 2 X 10(-8) M, and 2 X 10(-9) M, and the mol of ligand bound/mol of receptor monomer were 2.17, 1.85, 0.9, and 1.0, respectively. We conclude that the cation-independent mannose 6-phosphate receptor has two mannose 6-phosphate-binding sites/polypeptide chain.