Enzymatic phosphorylation of lysosomal enzymes in the presence of UDP-N-acetylglucosamine. Absence of the activity in I-cell fibroblasts.
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Animals↗
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Secretion by mononuclear phagocytes of lysosomal hydrolases bearing ligands for the mannose-6-phosphate receptor system of fibroblasts: evidence for a second mechanism of spontaneous secretion?
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Acetylglucosaminidase↗
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.
Antibodies, Monoclonal↗
beta-Galactosidase from rat epididymal fluid is bound by a recognition site attached to membranes of the epididymis different from the phosphomannosyl receptor.
In order to know if the beta-galactosidase of the rat epididymal fluid, as other secreted acid hydrolases, carries a marker in its molecule, we studied the binding of this enzyme to cellular membranes of the epididymal tissue. The binding, like that mediated by the phosphomannosyl receptor, was saturable, did not require calcium, had a Kd in the nM range and was inhibited by phosphatase or metaperiodate treatment of the enzyme. However fructose 6-phosphate derivates were more effective competitive inhibitors than mannose 6-phosphate. The binding capacity of the membranes were extractable with Triton X-100 and incorporable into liposomes. Trypsin inhibited the binding capacity of Triton extracts but it did not affect the affinity of intact cellular membranes for beta-galactosidase. The results suggest that a phosphorylated carbohydrate of the enzyme is bound by a recognizing site of the cellular membranes different from the phosphomannosyl receptor.
Animals↗
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.
Carrier Proteins↗
Mannose 6-phosphate/insulin-like growth factor II receptor: distinct binding sites for mannose 6-phosphate and insulin-like growth factor II.
Pentamannosyl phosphate substituted bovine serum albumin (PMP-BSA) and insulin like growth factor II (IGF II) bind specifically to immobilized mannose 6-phosphate/insulin like growth factor II receptor. An excess of IGF II inhibited binding of PMP-BSA by less than or equal to 20%, and an excess of PMP-BSA inhibited binding of IGF II by less than or equal to 10%. Polyclonal antibodies against the receptor purified from human liver inhibited preferentially the binding of PMP-BSA, and a monocloncal antibody 2C2 inhibited only the binding of IGF II to the receptor. Similar results were obtained for binding of PMP-BSA and IGF II to human skin fibroblasts. These results suggest that the binding sites for mannose 6-phosphate and IGF II reside in different portions of the receptor.
Binding Sites↗
Inhibition by lysosomal enzymes and mannose-6-phosphate of the phosphorylation of the lysosomal enzyme binding receptor protein from monkey brain.
The lysosomal enzymes beta-glucuronidase and alpha-L-fucosidase and mannose-6-phosphate inhibited the phosphorylation of the lysosomal enzyme binding receptor protein prepared from monkey brain. Inhibition of both serine and tyrosine phosphorylation was observed. A non-lysosomal glycoprotein enzyme butyrylcholinesterase, mannose or glucose did not inhibit phosphorylation. Tyrosine phosphorylation of histone by the receptor protein was also inhibited by the lysosomal enzymes and mannose-6-phosphate.
Animals↗
Interactions of recombinant and platelet transforming growth factor-beta 1 precursor with the insulin-like growth factor II/mannose 6-phosphate receptor.
Recombinant transforming growth factor (TGF)-beta 1 precursor was recently found to contain mannose 6-phosphate (Purchio et al., 1988, J. Biol. Chem. 263, 14211-14215). In the present study, recombinant TGF-beta 1 precursor was shown to bind to the insulin-like growth factor (IGF)-II/mannose 6-phosphate (man6P) receptor on the plasma membrane of cells since: 1) Insulin, which induces an increase in cell surface IGF-II/man6P receptors on adipocytes, caused a 2.7-fold increase in TGF-beta 1 precursor binding to adipocytes; 2) Chinese hamster ovary cells selected for overexpression of the IGF-II/man6P receptor exhibited an increased binding of TGF-beta 1 precursor in comparison to the parental cells; and 3) the binding of 125I-TGF-beta 1 precursor to these transfected cells and adipocytes was largely inhibited by man6P. After 15 minutes at 37 degrees C, 75% of the recombinant TGF-beta 1 precursor was found to be internalized in the transfected cells. Additional studies with latent TGF-beta 1 isolated from platelets indicated that this material could also bind to the isolated IGF-II/man6P receptor.
Adipose Tissue↗
Developmental regulation of insulin-like growth factor-II/mannose 6-phosphate receptor mRNA in the rat.
This study examined levels of insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/M6PR) mRNA in tissues of rats at different stages of growth. Northern blot analysis of total RNA from tissues of rats aged 2, 9, 21 and 42 days and from 21 day fetal rats was carried out using a cDNA probe to the IGF-II/M6PR. Northern blots showed this probe hybridized to a single 9kb band in all tissues tested. Highest hybridization signals were detected in fetal and neonatal tissues with levels rapidly decreasing after birth. For all age groups tested the highest signal was obtained with heart followed by muscle, lung, and kidney, with liver and brain showing lower levels of message. These results indicate that IGF-II/M6PR mRNA is developmentally regulated, and suggest a role for the IGF-II/M6PR in fetal and neonatal growth.
Aging↗
Insulin-like growth factor-II/mannose 6-phosphate receptor is incapable of activating GTP-binding proteins in response to mannose 6-phosphate, but capable in response to insulin-like growth factor-II.
We previously reported that insulin-like growth factor-II (IGF-II) stimulates both calcium influx and DNA synthesis by acting on the cell surface IGF-II receptor (IGF-IIR) in a manner sensitive to pertussis toxin, and recently demonstrated that IGF-II binding to the IGF-IIR gives rise to functional changes of purified Gi-2, a GTP-binding protein (G protein) in phospholipid vesicles as well as in broken cell membranes. On the other hand, a variety of evidence indicates that the IGF-IIR binds mannose 6-phosphate (man6P) with high affinity probably at a receptor extracellular region different from the IGF-II-binding site. In the present study, we examined whether man6P stimulation of the IGF-IIR evokes the activation of Gi-2 in phospholipid vesicles and in native cell membranes. In vesicles reconstituted with purified rat IGF-IIR and bovine Gi-2, man6P did not stimulate GDP dissociation from Gi-2 even in concentrations up to 10 mM, while IGF-II dose-dependently facilitated GDP release from Gi-2 with an EC50 of 6 nM. The stimulatory effect of IGF-II was not observed in vesicles reconstituted with Gi-2 alone. In addition, also in a native environment of cell membranes, man6P did not affect an endogenous 40-kDa protein or exogenously added purified Gi-2 as assessed with reduction of the pertussis toxin-catalyzed ADP-ribosylation. These results indicate that the IGF-IIR does not activate Gi-like proteins upon man6P binding in phospholipid vesicles and in native cellular membranes, whereas the receptor activates Gi-like proteins upon IGF-II binding in both environments. Thus, we postulate that the IGF-IIR dissimilarly responds to the two structurally unrelated ligands, IGF-II and man6P, in the linkage function with G proteins.
Animals↗
Insulin-like growth factor II overexpression does not affect sorting of lysosomal enzymes in NIH-3T3 cells.
The sorting of newly synthesized mannose 6-phosphate (M6P)-containing proteins and of the major excreted protein (MEP), a lysosomal thiol proteinase, was studied in NIH-3T3 cells transfected with the cDNA of human insulin-like growth factor II (IGF II) or with the vector alone. Extracts from media and cells labelled with [35S] methionine were used for chromatography on a M6P/IGF II receptor affinity matrix or for immunoprecipitation to assess the distribution of newly synthesized M6P-containing proteins and MEP, respectively. The results indicate that the overexpression of IGF II did not affect the synthesis and the sorting of M6P-containing proteins and of MEP. The binding and uptake of the lysosomal enzyme arylsulfatase A were not affected in IGF II overexpressing cells.
Animals↗
Brefeldin A affects the cellular distribution of endocytic receptors differentially.
The cell surface expression of three endocytic receptors was studied in human hepatoma Hep G2 cells treated with brefeldin A (BFA). Ligand binding and cell surface iodination revealed that BFA increased the number of mannose 6-phosphate/insulin-like growth factor II receptors twofold and decreased the amount of asialoglycoprotein and transferrin receptors by 40-60%. The altered expression of receptors at the cell surface was paralleled by changes in the respective ligand uptake. The implications of this finding on our understanding of intracellular trafficking are discussed.
Asialoglycoprotein Receptor↗
Mannose 6-phosphate potentiates insulin-like growth factor II effects in cultured human neuroblastoma cells.
Insulin-like growth factor II (IGF-II) and mannose 6-phosphate (man-6-P) bind to distinct sites on the same receptor. In the present study, we examined the effects of man-6-P on the growth promoting effects of IGF-II on SH-SY5Y cultured human neuroblastoma cells. Man-6-P alone increased cell number and neurite outgrowth by approximately 50%; as previously observed, IGF-II increased cell number and neurite outgrowth by approximately 110 and 30%, respectively. However, when cells were grown in the presence of both ligands, cell number increased by 330% and neurite outgrowth by 130%. These results suggest that man-6-P can potentiate the known growth promoting effects of IGF-II on human neuroblastoma cells. Furthermore, they indicate that the IGF-II/man-6-P receptor may serve as a means of integrating distinct growth promoting signals in neuronal cells.
Cell Division↗
Insulin and IGF-II, but not IGF-I, stimulate the in vitro regeneration of adult frog sciatic sensory axons.
We used the in vitro regenerating frog sciatic nerve to look for effects of insulin and insulin-like growth factors I and II (IGF-I, IGF-II) on regeneration of sensory axons and on injury induced support cell proliferation in the outgrowth region. In nerves cultured for 11 days, a physiological dose (10 ng/ml, approximately 2 nM) of insulin or IGF-II increased ganglionic protein synthesis (by 20% and 50%, respectively) as well as the level of newly formed, radiolabelled axonal material distal to a crush injury (both by 80%), compared to untreated, paired controls. In addition, insulin increased the outgrowth distance of the furthest regenerating sensory axons by 10%. The preparation was particularly sensitive to insulin during the first 5 days of culturing. Furthermore, both insulin and IGF-II were found to inhibit proliferation of support cells in the outgrowth region in a manner suggesting effects via their individual receptors. The inhibition, about 30%, was observable after 4 but not 11 days in culture. It is not clear if this reflects a stimulated differentiation of some cells. By contrast, IGF-I lacked effects on both regeneration and proliferation. In conclusion, the results suggest that insulin and IGF-II are involved in the regulation of peripheral nerve regeneration.
Animals↗
Synthesis of some D-mannosyl-disaccharides containing D-mannose 6-phosphate residues.
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Carbohydrate Conformation↗
Isolation and characterization of the 36-kDa D-mannose 6-phosphate receptor from porcine testis.
A D-mannose 6-phosphate receptor was isolated from total membranes of porcine testis, and its interaction with ligands was examined. The receptor was a glycoprotein comprised of several 36-kDa sub-units with an isoelectric point (pI) of 6.1. The binding of the receptor to the insoluble phosphomannan core occurred in the absence of divalent cations, but was selectively stimulated by MnCl2 and effectively inhibited by D-mannose 6-phosphate, D-fructose 1-phosphate, and pentamannosyl monophosphate. The phosphate group and HO-2 of D-mannose 6-phosphate are important in the receptor-ligand interaction, HO-4 probably contributes to a lesser extent, and HO-1 seems to have no interaction.
Animals↗
Preparation and application of a pentamannosyl monophosphate-bovine serum albumin conjugate.
Pentamannosyl monophosphate, derived from Hansenula holstii O-phosphomannan, was conjugated to bovine serum albumin by reductive amination. The conjugate inhibited the binding of the porcine testis mannose 6-phosphate receptor to the insoluble phosphomannan core. A mannose 6-phosphate receptor with a molecular weight of 200,000 was purified from porcine liver membranes, using an affinity matrix of the conjugate attached to Sepharose 4B. Rabbits were immunised with the conjugate, and the antisera were purified on a phosphomannan core-Sepharose 4B column in order to give an antibody which was specific for the 6-phosphate group and the equatorial HO-4 of D-mannose 6-phosphate. On Western blot analysis using the purified antibodies, ovalbumin, which contained a typical high-mannose type of oligosaccharide, was not recognised. However, a testicular glycoprotein fraction formed an immunostaining band. These results indicate the effectiveness of the conjugate as a ligand for mannose 6-phosphate receptors. The antibodies highly specific for mannose 6-phosphate may be used to detect or purify lysosomal enzymes.
Animals↗
Synthesis of a branched glycopeptide derivative containing terminal D-mannose 6-phosphate residues.
A branched glycopeptide derivative incorporating two D-mannose 6-phosphate residues was prepared by coupling 6-aminohexyl 6-O-[bis(2,2,2-trichloroethoxy)phosphinyl]-alpha-D-mannopyranoside with N-acetyl-L-tyrosyl-L-aspart-oyldi-L-alanine followed by reductive deprotection of the phosphate group.
Carbohydrate Conformation↗