Identification of residues essential for carbohydrate recognition and cation dependence of the 46-kDa mannose 6-phosphate receptor

The 46 kDa cation-dependent mannose 6-phosphate receptor (CD-MPR) plays an essential role in the biogenesis of lysosomes by diverting newly synthesized mannose 6-phosphate (Man-6-P)-containing lysosomal enzymes from the secretory pathway to acidified endosomes. Previous crystallographic studies of the CD-MPR have identified 11 amino acids within its carbohydrate binding pocket. These residues were evaluated quantitatively by assaying the binding affinity of mutant receptors containing a single amino acid substitution toward a lysosomal enzyme. The results show that substitution of Gln-66, Arg-111, Glu-133, or Tyr-143 results in a >800-fold decrease in affinity, demonstrating these four amino acids are essential for carbohydrate recognition by the CD-MPR. Solution binding and surface plasmon resonance analyses demonstrated that the presence of Mn2+ enhanced the affinity of the CD-MPR for a lysosomal enzyme by 2- to 4-fold and increased the stoichiometry of the interaction between a heterogeneous population of a lysosomal enzyme and the receptor by approximately 3-fold. In contrast, substitution of Asp-103 results in a protein that no longer exhibits enhanced binding affinities or altered stoichiometry in the presence of cations, and electron spin resonance demonstrated that the D103S mutant exhibits a 6-fold lower affinity for Mn2+ than the wild-type receptor (Kd = 3.7 6 1.4 mM versus 0.6 6 0.1 mM). Chemical cross-linking revealed that Mn2+ influences the stoichiometry of interaction between the CD-MPR and lysosomal enzymes by increasing the oligomeric state of the receptor from dimer to higher order oligomers. Taken together, these studies provide the molecular basis for high affinity carbohydrate recognition by the CD-MPR. Furthermore, Asp-103 has been identified as the key residue which mediates the effects of divalent cations on the binding properties of the CD-MPR.     

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.     

3-Methyladenine specifically inhibits retrograde transport of cation-independent mannose 6-phosphate/insulin-like growth factor II receptor from the early endosome to the TGN

3-Methyladenine (3-MA), a well-known inhibitor of autophagic sequestration, can also prevent class III phosphatidylinositide (PI) 3-kinase activity, which is required for many processes in endosomal membrane trafficking. Although much is known about the effects of other PI 3-kinase inhibitors, such as wortmannin and LY294002, on endosomal membrane trafficking, little is known about those of 3-MA. Here we show that the treatment of cells with 3-MA results in a specific redistribution of the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (MPR300) from the trans-Golgi network (TGN) to early/recycling endosomal compartments containing internalized transferrin. Importantly, in contrast to wortmannin and LY294002, 3-MA did not cause the enlargement of late endosomal/lysosomal compartments. The results suggest that the effect of 3-MA is restricted to the retrieval of MPR300 from early/recycling endosomes.     

Structural features of the lysosomal hydrolase mannose 6-phosphate uncovering enzyme

The uncovering enzyme (UCE) removes N-acetylglucosamine from lysosomal enzymes to uncover the mannose 6-phosphate (Man-6-P) determinant necessary for targeting these enzymes to lysosomes. Failure to create the Man-6-P determinant is one cause of lysosomal storage diseases. Despite its medical importance, little structural information about UCE is available. In this report we have developed a model for the membrane proximal portion of the lumenal domain of UCE based on the structure of the EFG-3 and -4 domains of the extracellular segment of the beta chain of integrin V 3. In this model the EGF-like domains of UCE (residues 285–345) are predicted to form a rod-shaped stalk region, similar to the stem region in Golgi glycosyltransferases. This stalk causes the proposed catalytic domain (residues 1–277) to be extended away from the Golgi membrane. A portion of the proposed catalytic domain (residues 85-256) resides in Cluster of Orthologous Group (COG) 4632 with four bacterial proteins but is not homologous to any known eukaryotic proteins. Thus, UCE may have evolved from the fusion of a unique catalytic domain with a common EGF-like stalk domain. We have determined by mass spectrometry that the four disulfide bonds of the proposed catalytic domain are located between Cys²–Cys¹⁷², Cys⁶⁶–Cys⁹⁹, Cys⁸³–Cys²⁷⁴, and Cys²⁵⁸–Cys²⁶⁵. Finally, we determined that four of the six potential N-linked glycosylation sites are glycosylated (Asn 159, Asn 165, Asn 247, and Asn 317) in COS cells. Published in 2005.     

Fibroblast chemotaxis and prolidase activity modulation by insulin-like growth factor II and mannose 6-phosphate

Chemotactic locomotion of fibroblasts requires extensive degradation of extracellular matrix components. The degradation is provided by a variety of proteases, including lysosomal enzymes. The process is regulated by cytokines. The present study shows that mannose 6-phosphate and insulin-like growth factor II (IGF-II) enhance fibroblast chemotaxis toward platelet-derived growth factor (PDGF). It is suggested that lysosomal enzymes (bearing mannose 6-phosphate molecules) are involved in chemotactic activity of the cells. The suggestion is supported by the observation that a-mannosidase and cathepsin D inhibitor - pepstatin are very potent inhibitors of fibroblast chemotaxis. Simultaneously, mannose 6-phosphate stimulates extracellular collagen degradation. The final step in collagen degradation is catalyzed by the cytosolic enzyme - prolidase. It has been found that mannose 6-phosphate stimulates also fibroblast prolidase activity with a concomitant increase in lysosomal enzymes activity. The present study demonstrates that the prolidase activity in fibroblasts may reflect the chemotactic activity of the cells and suggests that the mechanism of cell locomotion may involve lysosomal enzyme targeting, probably through IGF-II/mannose 6-phosphate receptor.     

The insulin-like growth factor II/mannose-6-phosphate receptor

Recent evidence from molecular cloning, biochemical and immunological experiments has established that the cation-independent mannose-6-phosphate (Man-6-P) receptor and insulin-like growth factor-II (IGF-II) receptor are the same protein. Although the role of the IGF-II/Man-6-P receptor as a transporter of hydrolytic enzymes in the biogenesis of lysosomes is certain, elucidation of the receptor's structure has not yet provided major insights into the function of IGF-II binding. Mutually exclusive binding of IGF-II and naturally occurring phosphomannosyl ligands to distinct but proximal sites on the receptor suggests that the IGF-II/Man-6-P receptor cannot simultaneously fulfill the functional requirements of both IGF-II and lysosomal enzymes. Does the receptor transduce on intracellular signal in order to mediate the biological effects of IGF-II? If so, then the receptor must interact with an effector molecule, perhaps a G protein, in the mechanism of IGF-II action. Further information from ligand binding and especially mutagenesis experiments will be needed to elucidate the potentially multiple functions of the IGF-II/Man-6-P receptor.     

Drug-induced phospholipidosis is caused by blockade of mannose 6-phosphate receptor-mediated targeting of lysosomal enzymes

Cationic amphiphilic drugs (CADs) cause massive intracellular accumulation of phospholipids, thereby resulting in phospholipidosis (PLD); however, the molecular mechanism underlying CAD-induced PLD remains to be resolved. Here, we found that treatment of normal rat kidney cells with CADs known to induce PLD caused redistribution of a mannose 6-phosphate/IGF-II receptor (MPR300) from the TGN to endosomes and concomitantly increased the secretion of lysosomal enzymes, resulting in a decline of intracellular lysosomal enzyme levels. These results enable the interpretation of why CADs cause excessive accumulation of undegraded substrates, including phospholipids in lysosomes, and led to the conclusion that the impaired MPR300-mediated sorting system of lysosomal enzymes reflects the general mechanism of CAD-induced PLD. In addition, our findings suggest that the measurement of lysosomal enzyme activity secreted into culture medium is useful as a rapid and convenient in vitro early screening system to predict drugs that can induce PLD.     

Developmental expression of the IGF-II/mannose 6-phosphate receptor

The first indication that the insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/M6PR) is developmentally regulated came from studies of the serum form of the receptor in the rat. By immunoblotting, the circulating form of the receptor, which was 10 kDa smaller than the tissue receptor, was high in 19 day fetal and 3, 10, and 20 day postnatal sera and then declined sharply. We next used quantitative immunoblotting to measure the total tissue IGF-II/M6PR in the rat. The receptor levels were high in fetal tissues and in most tissues declined dramatically in late gestation and/or in the early postnatal period. The rank order of receptor expression was heart > placenta > lung = intestine > muscle = kidney > liver > brain. In heart, the receptor was 1.7% of total protein in the extract. More recently, we have examined the expression of IGF-II/M6PR mRNA using Northern blotting and a solution hybridization/RNase protection assay. The rank order of receptor mRNA concentration among fetal tissues agreed with the rank order of receptor protein. The concentration of receptor mRNA was significantly lower in postnatal tissue than in fetal tissue. Thus IGF-II/M6PR mRNA concentration is an important determinant of receptor protein in most tissues. What is the function of the IGF-II/M6PR in embryonic and fetal tissues? The M6PR in birds and frogs does not bind IGF-II. It is intriguing that the rat IGF-II/M6PR is prominent during the embryonic and fetal periods, times at which the differences between mammals, on the one hand, and frogs and birds, on the other, are most striking. Tissue remodeling is an important feature of embryonic and fetal development. Therefore, the well-established lysosomal enzyme targeting function of the receptor may be of particular importance. Since IGF-II can inhibit the cellular uptake of lysosomal enzymes via the IGF-II/M6PR, IGF-II may modulate this lysosomal enzyme targeting function. In addition, the receptor can provide a degradative pathway for IGF-II by receptor-mediated internalization. Thus the receptor could provide a check on the high levels of IGF-II known to be present in the fetus. Finally, the IGF-II/M6PR could directly signal certain biologic responses to IGF-II. © 1993 Wiley-Liss, Inc.     

Biochemical and functional characterization of cation dependent (Mr 46,000) goat mannose 6-phosphate receptor

The Mannose 6-phosphate receptor (MPR’s) proteins are important for transporting lysosomal enzymes from trans-golgi to the pre-lysosomal compartment. These are conserved in the vertebrates from fish to mammals. We have cloned the full length cDNA for the goat MPR 46 protein and compared its sequences to the other known vertebrate MPR 46 proteins. In the present study the full-length cDNA for the goat MPR 46 protein was expressed in MPR deficient cells. The expressed protein was purified on the multivalent phosphomannan gel in the presence of divalent metal ions. The apparent molecular mass of the expressed protein was found to be ∼46 kDa and also exhibits oligomeric nature as observed in the other species, by using an MSC1 antibody (that recognizes the MPR 46 from molluscs to mammals) as well as with a peptide specific antibody corresponding to amino acid residues (218–237) of the cytoplasmic tail of human MPR 46 protein. Furthermore the distribution of the expressed protein was visualized by immunofluorescence using MSC1 and LAMP1 antibody. Additionally in the goat MPR 46 expressing cells, the sorting function of the expressed protein to sort cathepsin D to lysosomes was studied by confocal microscopy using cathepsin D antiserum and LAMP1 antibody. The binding of goat MPR 46 to cathepsin D was shown in far Western blotting and the mannose 6-phosphate dependent binding was shown by co-immunoprecipitation.     

Ile (476), a constituent of di-leucine-based motif of a major lysosomal membrane protein,LGP85/LIMP II,is important for its proper distribution in late endosomes and lysosomes

Lysosomal membrane glycoprotein termed LGP85 or LIMP II extends a COOH-terminal cytoplasmic tail of R459GQGSMDEGTADERAPLIRT478, in which an L475 I476 sequence lies as a di-leucine-based motif for lysosomal targeting. In the present study, we explored the role of the I476 residue in the localization of LGP85 to the endocytic organelles using two substitution mutants called I476A and I476L in which alanine and leucine are replaced at I476, respectively, and I476R477T478-deleted LGP85 called Delta 476-478. Immunofluorescence analyses showed that I476A and I476L are largely colocalized in intracellular organelles with an endogenous late endosomal and lysosomal marker, LAMP-1, but there were some granules in which staining for the LGP85 mutants was prominent, while Delta 476-478 is detected in LAMP-1-positive and LAMP-1-negative intracellular organelles, and on the cell surface. The subcellular fractionation studies revealed that I476A, I476L, and Delta 476-478 are different from wild-type LGP85 in the distribution of early endosomes, late endosomes, and lysosomes. I476A and I476L are present more in late endosomes than in the densest lysosomes, whereas wild-type LGP85 is mainly lysosomal. Substitution of I476 for A and L differentially modified the ratios of late endosomal to lysosomal LGP85. A major portion of Delta 476-478 resided in the light buoyant density fraction containing plasma membrane and early endosomes. Taken together, these results indicate that the existence of the 476th amino acid residue is essential for localization of LGP85 to late endocytic compartments. The fact that isoleucine but not leucine is in the 476th position is especially of importance in the proper distribution of LGP85 in late endosomes and lysosomes.     

Kinetics of insulin-like growth factor II (IGF-II) interaction with domain 11 of the human IGF-II/mannose 6-phosphate receptor: function of CD and AB loop solvent-exposed residues

Ligands of the IGF-II/mannose 6-phosphate receptor (IGF2R) include IGF-II and mannose 6-phosphate modified proteins. Disruption of the negative regulatory effects of IGF2R on IGF-II-induced growth can lead to embryonic lethality and cancer promotion. Of the 15 IGF2R extracellular domains, domains 1-3 and 11 are known to have a conserved beta-barrel structure similar to that of avidin and the cation-dependent mannose 6-phosphate receptor, yet only domain 11 binds IGF-II with high specificity and affinity. In order to define the functional basis of this critical biological interaction, we performed alanine mutagenesis of structurally determined solvent-exposed loop residues of the IGF-II-binding site of human domain 11, expressed these mutant forms in Pichia pastoris, and determined binding kinetics with human IGF-II using isothermal calorimetry and surface plasmon resonance with transition state thermodynamics. Two hydrophobic residues in the CD loop (F1567 and I1572) were essential for binding, with a further non-hydrophobic residue (T1570) that slows the dissociation rate. Aside from alanine mutations of AB loop residues that decrease affinity by modifying dissociation rates (e.g. Y1542), a novel mutation (E1544A) of the AB loop enhanced affinity by threefold compared to wild-type. Conversion from an acidic to a basic residue at this site (E1544K) results in a sixfold enhancement of affinity via modification principally of the association rate, with enhanced salt-dependence, decreased entropic barrier and retained specificity. These data suggest that a functional hydrophobic binding site core is formed by I1572 and F1567 located in the CD loop, which initially anchors IGF-II. Within the AB loop, residues normally act to either stabilise or function as negative regulators of the interaction. These findings have implications for the molecular architecture and evolution of the domain 11 IGF-II-binding site, and the potential interactions with other domains of IGF2R.     

The ubiquitin ligase RNF126 regulates the retrograde sorting of the cation-independent mannose 6-phosphate receptor

The ubiquitin proteasome system is central to the regulation of a number of intracellular sorting pathways in mammalian cells including quality control at the endoplasmic reticulum and the internalization and endosomal sorting of cell surface receptors. Here we describe that RNF126, an E3 ubiquitin ligase, is involved in the sorting of the cation-independent mannose 6-phosphate receptor (CI-MPR). In cells transiently depleted of RNF126, the CI-MPR is dispersed into Rab4 positive endosomes and the efficiency of retrograde sorting is delayed. Furthermore, the stable knockdown of RNF126 leads to the lysosomal degradation of CI-MPR and missorting of cathepsin D. RNF126 specifically regulates the sorting of the CI-MPR as other cargo that follow the retrograde sorting route including the cholera toxin, furin and TGN38 are unaffected in the absence of RNF126. Lastly we show that the RING finger domain of RNF126 is required to rescue the decrease in CI-MPR levels, suggesting that the ubiquitin ligase activity of RNF126 is required for CI-MPR sorting. Together, our data indicate that the ubiquitin ligase RNF126 has a role in the retrograde sorting of the CI-MPR     

Multiple routes of protein transport from endosomes to the trans Golgi network

Proteins use multiple routes for transport from endosomes to the Golgi complex. Shiga and cholera toxins and TGN38/46 are routed from early and recycling endosomes, while mannose 6-phosphate receptors are routed from late endosomes. The identification of distinct molecular requirements for each of these pathways makes it clear that mammalian cells have evolved more complex targeting mechanisms and routes than previously anticipated.     

A novel assay to demonstrate an intersection of the exocytic and endocytic pathways at early endosomes

The mechanism of transport of membrane proteins from the trans-Golgi to the cell surface is still poorly understood. Previous studies suggested that basolateral membrane proteins, such as the transferrin receptor and the asialoglycoprotein receptor H1, take an indirect route to the plasma membrane via an intracellular, most likely endosomal intermediate. To define this compartment we developed a biochemical assay based on the very definition of endosomes. The assay is based on internalizing anti-H1 antibodies via the endocytic cycle of the receptor itself. Internalized antibody formed immune complexes with newly synthesized H1, which had been pulse-labeled with [(35)S]sulfate and chased out of the trans-Golgi for a period of time that was insufficient for H1 to reach the surface. Hence, antibody capture occurred intracellularly. Double-immunofluorescence labeling demonstrated that antibody-containing compartments also contained transferrin and thus corresponded to early and recycling endosomes. The results therefore demonstrate an intracellular intersection of the exocytic and endocytic pathways with implications for basolateral sorting.     

Soluble CD26/dipeptidyl peptidase IV enhances transendothelial migration via its interaction with mannose 6-phosphate/insulin-like growth factor II receptor

CD26 is a T cell surface molecule with dipeptidyl peptidase IV (DPPIV) enzyme activity in its extracellular region. In addition to its membrane form, CD26 exists in plasma as a soluble form (sCD26), which is the extracellular domain of the molecule thought to be cleaved from the cell surface. In this paper, we demonstrate that sCD26 mediates enhanced transendothelial T cell migration, an effect that requires its intrinsic DPPIV enzyme activity. We also show that sCD26 directly targets endothelial cells and that mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGFIIR) on the endothelial cell surface acts as a receptor for sCD26. Our findings therefore suggest that sCD26 influences T cell migration through its interaction with M6P/IGFIIR.     

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.     

mVps24p functions in EGF receptor sorting/trafficking from the early endosome

Yeast Vps24p (vacuolar protein sorting) is part of a protein complex suggested to function in sorting/trafficking during endocytosis. We have characterized a mammalian homolog of the yeast protein, mVps24p, and examined its role in epidermal growth factor receptor trafficking. Endogenous mVps24p was distributed in both cytosol and in puncta and partially colocalized with markers for the trans-Golgi network. Adventitious expression of hrs or a mVps4p mutant deficient in ATPase activity caused a redistribution of both mVps24p and the M6PR to the resultant clustered/enlarged early endosomes. Expression of an mVps24p N-terminal fragment, that interacts with phosphatidylinositol 3,5-bisphosphate but not with mVps4p, produces enlarged early endosomes. More importantly, the mVps24p N-terminal fragment resulted in not only enhanced recycling, but also decreased degradation of the EGF receptor. These findings are consistent with a model in which mVps24p has a role in trafficking from the early endosome.     

The role of mannose-6-phosphate receptor and autophagy in influencing the outcome of combination therapy

Combining two different treatment modalities for targeting malignancies is gaining importance, with preclinical/clinical results indicating higher success rates in eradicating tumors or having longer remission periods. A better understanding of the synergy between the treatments helps in optimizing the dose and time of administration. We found that chemotherapy enhanced the levels of insulin-like growth factor 2 receptor/cation-independent mannose-6-phosphate receptor (IGF2R) on the surface of tumor cells, which leads to better tumor targeting by cytotoxic T cells (CTLs). Early evidence indicates that upregulation of IGF2R involves the autophagy pathway.     

The receptor recycling pathway contains two distinct populations of early endosomes with different sorting functions

Receptor recycling involves two endosome populations, peripheral early endosomes and perinuclear recycling endosomes. In polarized epithelial cells, either or both populations must be able to sort apical from basolateral proteins, returning each to its appropriate plasma membrane domain. However, neither the roles of early versus recycling endosomes in polarity nor their relationship to each other has been quantitatively evaluated. Using a combined morphological, biochemical, and kinetic approach, we found these two endosome populations to represent physically and functionally distinct compartments. Early and recycling endosomes were resolved on Optiprep gradients and shown to be differentially associated with rab4, rab11, and transferrin receptor; rab4 was enriched on early endosomes and at least partially depleted from recycling endosomes, with the opposite being true for rab11 and transferrin receptor. The two populations were also pharmacologically distinct, with AlF4 selectively blocking export of transferrin receptor from recycling endosomes to the basolateral plasma membrane. We applied these observations to a detailed kinetic analysis of transferrin and dimeric IgA recycling and transcytosis. The data from these experiments permitted the construction of a testable, mathematical model which enabled a dissection of the roles of early and recycling endosomes in polarized receptor transport. Contrary to expectations, the majority (>65%) of recycling to the basolateral surface is likely to occur from early endosomes, but with relatively little sorting of apical from basolateral proteins. Instead, more complete segregation of basolateral receptors from receptors intended for transcytosis occurred upon delivery to recycling endosomes.