An ELISA method to quantify the mannose 6-phosphate receptors

Mannose 6-phosphate receptor proteins mediate transport of lysosomal enzymes to lysosomes in eukaryotes. Two receptors designated as MPR 300 and MPR 46 based on their apparent molecular mass have been well studied from human and bovine liver. In humans, it has been shown that the receptors are present in different concentrations in different tissues. In the present study, MPR 300 and MPR 46 were purified from goat liver by phosphomannan affinity chromatography, and polyclonal antibodies were raised in rabbits. MPR 300 receptor specific antibodies have been purified from the antiserum using a goat-MPR 300-receptor gel. Using this affinity-purified antibody and the antiserum to goat MPR 46, as well as an affinity-purified MSC1 antibody that is specific for MPR 46, we developed an ELISA method to quantify both the receptors. The receptors could be measured in the concentration range of 1-10 ng using ELISA. The receptors could be quantified from membrane extracts of different tissues of goat and chicken using this method.     

Reptilian MPR 300 is also the IGF-IIR: Cloning, sequencing and functional characterization of the IGF-II binding domain

The mammalian cation-independent mannose 6-phosphate/insulin-like growth factor (IGF)-II receptor binds IGF-II with high affinity. Ligands transported by the MPR 300/IGF-IIR include IGF-II and mannose 6-phosphate-modified proteins. By targeting IGF-II to lysosomal degradation, it plays a key role in the maintenance of correct IGF-II levels in the circulation and in target tissues. Although, from our studies we found homologous receptor in calotes but its functional significance was not known. We present here the first report on the calotes MPR 300/IGF-IIR binds IGF-II with K_d of 12.02 nM; these findings provide new and strong evidence that MPR 300/IGF-IIR in Calotes versicolor binds IGFII with high affinity.     

Identification of the Putative Mannose 6-phosphate Receptor Protein (MPR 300) in the Invertebrate unio

In mammals, Mannose 6-phosphate receptor proteins (MPR 300 and MPR 46) mediate transport of lysosomal enzymes to lysosomes. Both receptors have been found in non-mammalian vertebrates including fish. To investigate the presence of MPRs in invertebrates, MPR 300 protein was isolated from the mollusc unio by affinity chromatography. It was shown to exhibit biochemical and immunological properties similar to mammalian MPR 300.     

P-type lectins

The two members of the P-type lectin family, the cation-dependent mannose 6-phosphate receptor (CD-MPR) and the insulin-like growth factor II/mannose 6-phosphate receptor (IGF-II/MPR), are distinguished from all other lectins by their ability to recognize phosphorylated mannose residues. The P-type lectins play an essential role in the generation of functional lysosomes within the cells of higher eukaryotes by directing newly synthesized lysosomal enzymes bearing the mannose 6-phosphate (M6P) signal to lysosomes. At the cell surface, the IGF-II/MPR also binds to the nonglycosylated polypeptide hormone, IGF-II, targeting this potent mitogenic factor for degradation in lysosomes. Moreover, in recent years, the multifunctional nature of the IGF-II/MPR has become increasingly apparent, as the list of extracellular ligands recognized by this receptor has grown to include a diverse spectrum of M6P-containing proteins as well as nonglycosylated ligands, implicating a role for the IGF-II/MPR in a number of important physiological pathways. Recent investigations have provided valuable insights into the molecular basis of ligand recognition by the MPRs as well as the complex intracellular trafficking pathways traversed by these receptors. This review provides a current view on the structures, functions, and medical relevance of the P-type lectins.     

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.     

The chicken liver cation-independent mannose 6-phosphate receptor lacks the high affinity binding site for insulin-like growth factor II

The chicken liver cation-independent mannose 6-phosphate receptor has been purified to apparent homogeneity by affinity chromatography on pentamannose phosphate-Sepharose and tested for its ability to bind iodinated human IGF-I, human IGF-II, and chicken IGF-II. In contrast to the bovine, rat, and human cation-independent mannose 6-phosphate receptors, which bind human IGF-II and IGF-I with nanomolar and micromolar affinities, respectively, the chicken receptor failed to bind either radioligand at receptor concentrations as high as 1 microM. The bovine receptor binds chicken IGF-II with high affinity while the chicken receptor binds this ligand with only low affinity, which we estimate to be in the micromolar range. These data demonstrate that the chicken cation-independent mannose 6-phosphate receptor lacks the high affinity binding site for IGF-II. These results provide an explanation for the failure of previous investigators to identify the type II IGF receptor by IGF-II cross-linking to chicken cells and indicate that the mitogenic activity of IGF-II in chick embryo fibroblasts is most likely mediated via the type I IGF receptor.     

Selective recycling of the mannose 6-phosphate/IGF-II receptor to the trans Golgi network in vitro

Mannose 6-phosphate receptors carry soluble lysosomal enzymes from the trans Golgi network (TGN) to prelysosomes, and then return to the TGN for another round of lysosomal enzyme sorting. We describe here a complementation scheme that detects the vesicular transport of the 300 kd mannose 6-phosphate/IGF-II receptor from prelysosomes to the TGN in cell extracts. In vitro transport displays the same selectivity observed in living cells in that the transferrin receptor traverses to the TGN at a much lower rate than mannose 6-phosphate receptors. Furthermore, recycling of mannose 6-phosphate/IGF-II receptors to the TGN requires GTP hydrolysis and can be distinguished biochemically from the constitutive transport of proteins between Golgi cisternae by its resistance to the weak base, primaquine.     

Interactions of the receptor for insulin-like growth factor II with mannose-6-phosphate and antibodies to the mannose-6-phosphate receptor

Recently, the sequence of the human receptor for insulin-like growth factor II (IGF-II) was found to be 80% identical [Morgan et al., (1987) Nature 329, 301-307] to the sequence of a partial clone of the bovine cation-independent mannose-6-phosphate receptor [Lobel et al., (1987) Proc. Natl. Acad. Sci. USA 84, 2233-2237]. In the present study, the purified receptor for insulin-like growth factor II (IGF-II) was found to react with two different polyclonal antibodies to the purified mannose-6-phosphate receptor. Moreover, mannose-6-phosphate was found to stimulate the binding of labeled IGF-II to the IGF-II receptor by two-fold. This effect had the same specificity and affinity as the reported binding of mannose-6-phosphate to its receptor; mannose-1-phosphate and mannose had no effect on the binding of labeled IGF-II to its receptor, and the half-maximally effective concentration of mannose-6-phosphate was 0.3 mM. Also, mannose-6-phosphate did not affect labeled IGF-II binding to the insulin receptor. These results support the hypothesis that a single protein of Mr-250,000 binds both IGF-II and mannose-6-phosphate. Furthermore, they indicate that mannose-6-phosphate can modulate the interaction of IGF-II to its receptor.     

Identification of the putative mannose 6-phosphate receptor (MPR 46) protein in the invertebrate mollusc

Mannose 6-phosphate receptor (MPR 300) protein was earlier affinity purified on phosphomannan gel from the membrane extracts of whole animal acetone powder of a mollusc, unio, in the presence of EDTA (Udaya Lakshmi, Y., Radha, Y., Hille-Rehfeld, A., von Figura, K., and Siva Kumar, N. (1999) Biosci. Rep. 19:403–409). In the present study we demonstrate that the unio also contains the putative mannose 6-phosphate receptor (MPR 46) that can be purified on the same gel in presence of divalent metal ions (10 mM each of calcium, manganese, and magnesium), and in the absence of sodium chloride and at pH 6.5. Chicken and Fish cell MPR 46 proteins were purified under these conditions (Siva Kumar, N., Udaya Lakshmi, Y., Hille-Rehfeld, A., and von Figura, K. (1999) Comp. Biochem. & Physiol. 123B:261–265). The authenticity of the receptor is further confirmed by its ability to react with the MSC1 antibody that is specific for MPR 46 protein. Additional evidence for the presence of MPR 46 in molluscs could be obtained by metabolic labeling of mollusc cells Biomphalaria glabrata (Bg cells) with [³⁵S] methionine and cysteine, and passing the labeled membrane extract on phosphomannan gel (at pH 6.5 and 7.0). On elution with mannose 6-phosphate, followed by immunoprecipitation of the column fractions, we identified the putative MPR 46 protein in the Bg cells. When Bg cell MPR 46 was deglycosylated along with chicken MPR 46 (control) both species yielded a single polypeptide corresponding to molecular mass of 26 kDa, suggesting that both contain the same receptor protein.     

Beta-galactosidase decreases the binding affinity of the insulin-like-growth-factor-II/mannose-6-phosphate receptor for insulin-like-growth-factor II

The insulin-like growth-factor-II/mannose-6-phosphate (IGF-II/Man6P) receptor binds two classes of ligands, insulin-like growth factors and lysosomal enzymes. We have examined the ability of the lysosomal enzyme, beta-galactosidase, to modulate the binding of 125I-IGF-II to the receptor. beta-Galactosidase purified from bovine testis was fractionated on a DEAF-Sephacel ion-exchange column. Column fractions were assayed for enzymatic activity and for ability to inhibit the binding of 125I-IGF-II to the IGF-II/Man6P receptor. Enzyme fractions eluting at higher NaCl concentrations which had previously been shown to exhibit greater uptake by cells in culture, exhibited greater potency in inhibiting the binding of 125I-IGF-II to the receptor. A pool of these fractions from the DEAE-Sephacel column inhibited 125I-IGF-II binding to pure receptor by 80% with the concentration required for half-maximal inhibition being 25 nM. The inhibition of binding by beta-galactosidase was completely blocked by simultaneous incubation with Man6P. Inhibition of the enzymatic activity of beta-galactosidase with D-galactonic acid gamma-lactone did not affect the ability of beta-galactosidase to inhibit the binding of 125I-IGF-II to the receptor. Scatchard analysis of IGF-II binding to pure receptor in the presence and absence of beta-galactosidase showed that beta-galactosidase decreased the binding affinity for IGF-II (Kd 0.26 nM versus 1.0 nM in the presence of 57 nM beta-galactosidase). We confirmed the observations of others that Man6P alone actually increases the binding of 125I-IGF-II to the IGF-II/Man6P receptor, but we found that this phenomenon was dependent upon the method of preparation of the IGF-II/Man6P receptor. Microsomal membrane preparations, solubilized membranes, and receptors purified on an IGF-II-Sepharose column all exhibited stimulation of 125I-IGF-II binding by Man6P, whereas receptors purified on lysosomal enzyme affinity columns showed little or no stimulation of 125I-IGF-II binding by Man6P. We conclude that beta-galactosidase decreases the binding affinity of the IGF-II/Man-6-P receptor for IGF-II by binding with high affinity to the Man6P-recognition site.     

Role of cation independent mannose 6-phosphate receptor protein in sorting and intracellular trafficking of lysosomal enzymes in chicken embryonic fibroblast (CEF) cells

Delivery of soluble lysosomal proteins to the lysosomes is dependent primarily on the mannose 6-phosphate receptors (MPRs) in mammals. However, in non-mammalian cells the role of MPR300 in sorting and trafficking of acid hydrolases to lysosomes is not fully understood till now. In this paper, we tested the role of MPR300 in sorting and trafficking of lysosomal enzymes in CEF cells using a small interfering RNA (siRNA) technology. Inactivation of MPR300 resulted in the secretion of large amounts of newly synthesized hydrolases into the medium and also inhibited the endocytosis of mannose 6-phospharylated ligands. Knockdown of MPR300 in CEF cells results in missorting of fucosidase and arylsulfatse A enzymes into the medium. The results indicated that the MPR300 in CEF cells plays a key role in sorting and trafficking of these soluble hydrolases.     

Molecular cloning, expression and functional characterization of the chicken cation dependent mannose 6-phosphate receptor protein

Mammalian mannose 6-phosphate (M6P) receptors function in transport of lysosomal enzymes. To understand the structural and functional significance of the chicken cation dependent mannose 6-phosphate receptor (MPR) (Mr 46kDa), a full-length cDNA for the chicken protein was cloned and expressed in mpr((-/-)) MEF cells devoid of both the receptors. The stably transfected cells express the receptor that could be affinity purified by phosphomannan chromatography. The authenticity of the receptor was confirmed by its immuno-reactivity with mammalian MPR 46 antibodies and its ability to sort cathepsin D in transfected cells (92.3%) as compared to mock transfected cells (50.2%), establishing a functional role for the chicken receptor.     

Mannose 6-Phosphate Receptor Proteins from Reptiles and Amphibians: Evidence for the Presence of MPR 300 and MPR 46

Two mannose 6-phosphate receptors (MPR 300 and MPR 46) are involved in transport of lysosomal enzymes. Both receptors are expressed in all mammalian species studied so far and in chicken. Here we present the first report on affinity purification of both MPRs from the liver tissues of reptiles and amphibians using Sepharose divinyl sulfone phosphomannan at pH 7.0. MPR 300 from both species show similar electrophoretic mobility as mammalian MPR 300 and cross-react with an antibody directed against MPR 300 from goat liver. Furthermore, MPR 46 from reptilian liver and amphibian oocytes cross-react with peptide-specific antibodies against the cytoplasmic domain of human MPR 46 (anti-MSC1).     

Mannose-6-phosphate receptors (MPR 300 and 46) from the highly evolved invertebrate <Emphasis Type="Italic">Asterias rubens</Emphasis> (Echinodermate): biochemical and functional characterization of MPR 46 protein

Mammalian mannose 6-phosphate receptors (MPR 300 and 46) mediate transport of lysosomal enzymes to lysosomes. Recent studies established that the receptors are conserved throughout vertebrates. Although we purified the mollusc receptors and identified only a lysosomal enzyme receptor protein (LERP) in the Drosophila melanogaster, little is known about their structure and functional roles in the invertebrates. In the present study, we purified the putative receptors from the highly evolved invertebrate, starfish, cloned the cDNA for the MPR 46, and expressed it in mpr^(−/−) mouse embryonic fibroblast cells. Structural comparison of starfish receptor sequences with other vertebrate receptors gave valuable information on its extensive structural homology with the vertebrate MPR 46 proteins. The expressed protein efficiently sorts lysosomal enzymes within the cells establishing a functional role for this protein. This first report on the invertebrate MPR 46 further confirms the structural and functional conservation of the receptor not only in the vertebrates but also in the invertebrates.     

Targeted disruption of the M(r) 46,000 mannose 6-phosphate receptor gene in mice results in misrouting of lysosomal proteins.

Lysosomal enzymes containing mannose 6-phosphate recognition markers are sorted to lysosomes by mannose 6-phosphate receptors (MPRs). The physiological importance of this targeting mechanism is illustrated by I-cell disease, a fatal lysosomal storage disorder caused by the absence of mannose 6-phosphate residues in lysosomal enzymes. Most mammalian cells express two MPRs. Although the binding specificities, subcellular distribution and expression pattern of the two receptors can be differentiated, their coexpression is not understood. The larger of the two receptors with an M(r) of approximately 300,000 (MPR300), which also binds IGFII, appears to have a dominant role in lysosomal enzyme targeting, while the function of the smaller receptor with an M(r) of 46,000 (MPR46) is less clear. To investigate the in vivo function of the MPR46, we generated MPR46-deficient mice using gene targeting in embryonic stem cells. Reduced intracellular retention of newly synthesized lysosomal proteins in cells from MPR46 -/- mice demonstrated an essential sorting function of MPR46. The phenotype of MPR46 -/- mice was normal, indicating mechanisms that compensate the MPR46 deficiency in vivo.     

I-cell disease-like phenotype in mice deficient in mannose 6-phosphate receptors

Mannose 6-phosphate receptor deficient mice were generated by crossing mice carrying null alleles for Igf2 and the 300 kDa and 46 kDa mannose 6-phosphate receptors, Mpr300 and Mpr46. Pre- and perinatal lethality of mice nullizygous for Igf2, Mpr300 and Mpr46 was increased. Triple deficient mice surviving the first postnatal day had normal viability and developed a phenotype resembling human I-cell disease. The triple deficient mice were characterized by dwarfism, facial dysplasia, waddling gait, dysostosis multiplex, elevated lysosomal enzymes in serum and histological signs of lysosomal storage predominantly in fibroblasts, but also in parenchymal cells of brain and liver. A paternally inherited Mpr300 wild type allele that is normally inactive in mice due to imprinting was reactivated in some tissues of mice lacking IGF II and MPR 46 and carrying a maternal Mpr300 null allele. Inspite of the partial reactivation the phenotype of these mice was similar to that of triple deficient mice.     

Chicken and Xenopus mannose 6-phosphate receptors fail to bind insulin-like growth factor II

The recent demonstration that a single mammalian receptor protein binds both mannose 6-phosphate (Man-6-P) and insulin-like growth factor II (IGF-II) with high affinity has suggested a multifunctional physiological role for this receptor, possibly including signal transduction. In order to better understand the functions of this receptor, we have investigated the properties of Man-6-P receptors from non-mammalian species. Receptors were affinity-purified from Triton X-100 extracts of total membranes from Xenopus and chicken liver as well as rat placenta using pentamannosyl 6-phosphate-Sepharose. The Man-6-P receptor was adsorbed to the pentamannosyl 6-phosphate-Sepharose and specifically eluted by Man-6-P in all three species, as evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining. When the purified receptors from these three species were cross-linked to 125I-IGF-II with disuccinimidyl suberate, only receptors isolated from rat membranes were affinity-labeled. To further evaluate the properties of these Man-6-P receptors, binding of 125I-rat-IGF-II and 125I-chicken Tyr-Gly-Thr-Ala-IGF-II to purified receptors from Xenopus, chicken, and rat was evaluated by polyethylene glycol precipitation. Only the rat Man-6-P receptor exhibited detectable binding of 125I-IGF-II. These data suggest that the emergence of a high affinity IGF-II binding site on the Man-6-P receptor occurred in evolution after the divergence of mammals from other vertebrates. Thus, the biological actions of IGF-II in chickens and frogs appear to be initiated by the type I IGF receptor.