Mannose 6-phosphate Recognition Marker Research Articles

Human Mannose 6-Phosphate-uncovering Enzyme Is Synthesized as a Proenzyme That Is Activated by the Endoprotease Furin

N-Acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase, also known as "uncovering" enzyme (UCE), is localized in the trans-Golgi network, where it removes a covering N-acetylglucosamine from the mannose 6-phosphate recognition marker on lysosomal acid hydrolases. Here we show that UCE is synthesized as an inactive proenzyme that is activated by the endoprotease furin, which cleaves an RARLPR/D sequence to release a 24-amino acid propiece. As furin is localized in the trans-Golgi network, newly synthesized UCE is inactive until it reaches this terminal Golgi compartment. LoVo cells (derived from a human colon adenocarcinoma) lack furin activity and have extremely low UCE activity. Addition of furin to LoVo cell extracts restores UCE activity to normal levels, demonstrating that the UCE proenzyme is stable in this cell type. LoVo cells secrete acid hydrolases with phosphomannose diesters as a consequence of the deficient UCE activity. This demonstrates for the first time that UCE is the only enzyme in these cells capable of efficiently uncovering phosphomannose diesters. UCE also hydrolyzes UDP-GlcNAc, a sugar donor for Golgi N-acetylglucosaminyltransferases. The fact that UCE is not activated until it reaches the trans-Golgi network may ensure that the pool of UDP-GlcNAc in the Golgi stack is not depleted, thereby maintaining proper oligosaccharide assembly.

Multiple Signals Regulate Trafficking of the Mannose 6-Phosphate-uncovering Enzyme

The "uncovering enzyme," which catalyzes the second step in the formation of the mannose 6-phosphate recognition marker on lysosomal enzyme oligosaccharides, resides primarily in the trans-Golgi network and cycles between this compartment and the plasma membrane. An analysis of green fluorescent protein-uncovering enzyme chimeras revealed that the transmembrane segment and the first 11 residues of the 41-residue-cytoplasmic tail are sufficient for retention in the trans-Golgi network. The next eight residues ((486)YAYHPLQE(493)) facilitate exit from this compartment. Kinetic studies demonstrated that the (488)YHPL(491) sequence also mediates rapid internalization at the plasma membrane. This motif binds adaptor protein-2 in glutathione S-transferase-uncovering enzyme-cytoplasmic tail pull-down assays, indicating that the uncovering enzyme is endocytosed via clathrin-coated vesicles. Consistent with this finding, endogenous uncovering enzyme was detected in purified clathrin-coated vesicles. The enzyme with a Y486A mutation is internalized normally but accumulates on the cell surface because of increased recycling to the plasma membrane. This residue is required for efficient return of the enzyme from endosomes to the trans-Golgi network. These findings indicate that the YAYHPLQE motif is recognized at several sorting sites, including the trans-Golgi network, the plasma membrane, and the endosome.

Lysosomal Hydrolase Mannose 6-Phosphate Uncovering Enzyme Resides in thetrans-Golgi Network

A crucial step in lysosomal biogenesis is catalyzed by "uncovering" enzyme (UCE), which removes a covering N-acetylglucosamine from the mannose 6-phosphate (Man-6-P) recognition marker on lysosomal hydrolases. This study shows that UCE resides in the trans-Golgi network (TGN) and cycles between the TGN and plasma membrane. The cytosolic domain of UCE contains two potential endocytosis motifs: (488)YHPL and C-terminal (511)NPFKD. YHPL is shown to be the more potent of the two in retrieval of UCE from the plasma membrane. A green-fluorescent protein-UCE transmembrane-cytosolic domain fusion protein colocalizes with TGN 46, as does endogenous UCE in HeLa cells, showing that the transmembrane and cytosolic domains determine intracellular location. These data imply that the Man-6-P recognition marker is formed in the TGN, the compartment where Man-6-P receptors bind cargo and are packaged into clathrin-coated vesicles.

Azurophilic Granules of Human Neutrophilic Leukocytes Are Deficient in Lysosome-Associated Membrane Proteins but Retain the Mannose 6-Phosphate Recognition Marker

During granulocyte differentiation in the bone marrow (BM), neutrophilic leukocyte precursors synthesize large amounts of lysosomal enzymes. These enzymes are sequestered into azurophilic storage granules until used days later for digestion of phagocytized microorganisms after leukocyte emigration to inflamed tissues. This azurophil granule population has previously been defined as a primary lysosome, ie, a membrane-bound organelle containing acid hydrolases that have not entered into a digestive event. In this study, azurophil granules were purified and shown to contain large amounts of mannose 6-phosphate-containing glycoproteins (Man 6-P GP) but little lysosome-associated membrane proteins (LAMP). In addition, the fine structural localization of Man 6-P GP and LAMP was investigated at various stages of maturation in human BM and blood. Man 6-P GP were present within the azurophilic granules at all stages of maturation and in typical multivesicular bodies (MVB) as well as in multilaminar compartments (MLC), identified by their content of concentric arrays of internal membranes. LAMP was absent in all identified granule populations, but was consistently found in the membranes of vesicles, MVB, and MLC. The latter compartment has not been previously described in this cell type. In conclusion, the azurophilic granules, which contain an abundance of lysosomal enzymes and Man 6-P GP, lack the LAMP glycoproteins. By current criteria, they therefore cannot be classified as lysosomes, but rather may have the functional characteristics of a regulated secretory granule. Rather, the true lysosomes of the resting neutrophil are probably the MVB and MLC. Finally, the typical “dense bodies” or mature lysosomes described in other cells are not present in resting neutrophils.

Azurophilic Granules of Human Neutrophilic Leukocytes Are Deficient in Lysosome-Associated Membrane Proteins but Retain the Mannose 6-Phosphate Recognition Marker

AbstractDuring granulocyte differentiation in the bone marrow (BM), neutrophilic leukocyte precursors synthesize large amounts of lysosomal enzymes. These enzymes are sequestered into azurophilic storage granules until used days later for digestion of phagocytized microorganisms after leukocyte emigration to inflamed tissues. This azurophil granule population has previously been defined as a primary lysosome, ie, a membrane-bound organelle containing acid hydrolases that have not entered into a digestive event. In this study, azurophil granules were purified and shown to contain large amounts of mannose 6-phosphate-containing glycoproteins (Man 6-P GP) but little lysosome-associated membrane proteins (LAMP). In addition, the fine structural localization of Man 6-P GP and LAMP was investigated at various stages of maturation in human BM and blood. Man 6-P GP were present within the azurophilic granules at all stages of maturation and in typical multivesicular bodies (MVB) as well as in multilaminar compartments (MLC), identified by their content of concentric arrays of internal membranes. LAMP was absent in all identified granule populations, but was consistently found in the membranes of vesicles, MVB, and MLC. The latter compartment has not been previously described in this cell type. In conclusion, the azurophilic granules, which contain an abundance of lysosomal enzymes and Man 6-P GP, lack the LAMP glycoproteins. By current criteria, they therefore cannot be classified as lysosomes, but rather may have the functional characteristics of a regulated secretory granule. Rather, the true lysosomes of the resting neutrophil are probably the MVB and MLC. Finally, the typical “dense bodies” or mature lysosomes described in other cells are not present in resting neutrophils.

Re-expression of the Mannose 6-Phosphate Receptors in Receptor-deficient Fibroblasts: COMPLEMENTARY FUNCTION OF THE TWO MANNOSE 6-PHOSPHATE RECEPTORS IN LYSOSOMAL ENZYME TARGETING

We have previously generated primary embryonic fibroblasts lacking either the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (MPR) or the cation-dependent MPR, two trans-membrane proteins that bind the mannose 6-phosphate (Man-6-P) recognition marker on soluble lysosomal enzymes (Ludwig, T., Munier-Lehmann, H., Bauer, U., Hollinshead, M., Ovitt, C., Lobel, P., and Hoflack, B.(1994) EMBO J. 13, 3430-3437). These two cell types partially missort phosphorylated lysosomal enzymes. Using two-dimensional gel electrophoresis, we show here that they secrete, in a large part, different phosphorylated ligands. In order to better understand the sorting function of the MPRs, we have re-expressed each MPR in MPR-negative fibroblasts. We show that the MPRs have similar capacities for transporting the bulk of the newly synthesized lysosomal enzymes and that they target individual ligands with various efficiencies. However, high levels of one MPR do not fully compensate for the absence of the other, demonstrating that the two MPRs have complementary targeting functions, perhaps by recognizing different features on lysosomal enzymes. The analysis of the phosphorylated oligosaccharides shows that the ligands missorted in the absence of the cation-dependent MPR are slightly but significantly depleted in oligosaccharides with two Man-6-P residues, when compared with those missorted in the absence of the cation-independent MPR. While these results could explain some differences between the structure and the sorting function of the two MPRs, they strongly suggest that the reason why cells express two different but related MPRs is to maintain an efficient Man-6-P-dependent targeting process that could be potentially regulated by MPR expression.

Control of insulin-like growth factor-II/mannose 6-phosphate receptor gene transcription by proximal promoter elements.

The insulin-like growth factor-II/cation-independent mannose 6-phosphate receptor (IGF-II/MPR) is a multifunctional protein that binds IGF-II and ligands containing a mannose 6-phosphate recognition marker. Recent studies have shown that this receptor plays a critical role in mammalian development, and that its expression is controlled by both epigenetic and tissue-specific factors. Our laboratory has cloned the 93-kilobase mouse gene and characterized its 48 exons. In this report we describe the structure and function of the IGF-II/MPR gene promoter. To study promoter function, a series of chimeric plasmids linking different segments of IGF-II/MPR 5' flanking DNA to the reporter gene, firefly luciferase, were transiently transfected into HepG2 and C3H 10T1/2 cells. Promoter activity was orientation-specific and was maximal (550- to 4250-fold above promoterless control) with a plasmid containing 266 base pairs (bp) of IGF-II/MPR DNA. The fusion gene accurately directed transcription as measured by ribonuclease protection assay using RNA extracted from transfected cells. DNA-protein binding studies by in vitro DNase I footprinting revealed an extended 54-bp footprint within the proximal promoter that contained two E-boxes and potential binding sites for transcription factors Sp1, NGF-IA, and related proteins. Gel mobility shift experiments with double-stranded oligonucleotides containing this region gave rise to several specific DNA-protein complexes, and the addition of specific antibodies indicated that proteins antigenically related to Sp1 and c-Myc were components of one or more of these bands. Deletion of this 54-bp segment led to an 8-fold decline in promoter activity, and its transfer to a heterologous promoter stimulated gene expression by nearly 7-fold. Mutational analyses indicated that each E box contributed to more than half of the enhancer's activity. These results define a strong minimal IGF-II/MPR promoter of no more than 266 bp and identify a 54-bp enhancer within this promoter fragment. Our observations thus represent a first step toward characterizing the developmental, epigenetic, and tissue-specific factors that control IGF-II/MPR gene expression.

Does the overexpression of pro-insulin-like growth factor-II in transfected human embryonic kidney fibroblasts increase the secretion of lysosomal enzymes?

Insulin-like growth factor-II (IGF-II) and lysosomal enzymes bearing the mannose 6-phosphate (Man6P) recognition marker, bind to two distinct binding sites of the IGF-II/M6P receptor. The two classes of ligands reciprocally modulate the binding of the other class of ligand to the receptor [Kiess, W., Thomas, C. L., Greenstein, L., Lee, L., Sklar, M. M., Rechler, M. M., Sahagian, G. G. & Nissley, S. P. (1989) J. Biol. Chem. 264, 4710-4714]. We asked whether or not overexpression of pro-IGF-II by cells in culture leads to missorting of lysosomal enzymes. Human embryonal kidney fibroblasts were transfected with the full-length human IGF-II cDNA or a control cDNA. Solution hybridization/RNase protection experiments using a human IGF-II riboprobe showed that two transfectants expressed large quantities of IGF-II mRNA, whereas the non-transfected cells did not. The analysis of conditioned media revealed that these cells secrete approximately 0.15 micrograms and 1.0 micrograms immunoreactive IGF-II/ml and 22 x 10(6) cells and 24 x 10(6) cells within 24 hours. Immunoreactive IGF-II was shown by Western blotting to represent 17-kDa pro-IGF-II. The amount of the lysosomal enzyme, beta-hexosaminidase, was approximately twofold increased in the conditioned media from pro-IGF-II overexpressing cells compared with control media, as shown by Western-blot analysis and immunoprecipitation of media extracts of metabolically labeled cells. The synthesis rate of beta-hexosaminidase was not affected by pro-IGF-II overexpression. In addition, the basal amount of another newly synthesized lysosomal enzyme, the cathepsin D precursor, was also twofold higher in pro-IGF-II overexpressing cells than in control cells. In contrast, the surface binding and cellular uptake rate of a Man6P-containing neoglycoprotein did not differ between the cell lines. The results indicate that the overexpression of pro-IGF-II doubles the secretion and/or reduces the re-uptake of beta-hexosaminidase and cathepsin D to approximately 20% of the total synthesized enzymes in human embryonal kidney fibroblasts compared to control cells. We hypothesize that, in cells synthesizing high amounts of pro-IGF-II, the growth factor may modulate the targeting of a portion of lysosomal enzymes, mainly by partially enhancing the secretion of newly synthesized enzymes and, in addition, possibly by affecting the re-uptake mechanism.

Roles for mannose-6-phosphate receptors in lysosomal enzyme sorting, IGF-II binding and clathrin-coat assembly.

Two related transmembrane proteins in mammalian cells bind the mannose 6-phosphate recognition marker present on all soluble lysosomal hydrolases. Recent studies of cells that express only one or neither of these proteins have shed light not only on their function in directing lysosomal enzymes into the endocytic pathway but also on their critical role in transport vesicle formation in the trans Golgi network. One of these proteins also binds insulin-like growth factor II (IGF-II) and may be an important part of the IGF-dependent system that regulates development.

The Mouse Insulin-like Growth Factor II/Cation-Independent Mannose 6-Phosphate (IGF-II/MPR) Receptor Gene: Molecular Cloning and Genomic Organization

The mammalian insulin-like growth factor II/cation-independent mannose 6-phosphate receptor (IGF-II/MPR) is a multifunctional protein that binds both IGF-II and ligands containing a mannose 6-phosphate recognition marker through distinct high-affinity sites. This receptor plays an integral part in lysosomal enzyme transport, has a potential role in growth factor maturation and clearance, and may mediate IGF-II-activated signal transduction through a G-protein-coupled mechanism. Recent studies have shown that production of IGF-II/MPR mRNA and protein begins in the mouse embryo soon after fertilization and have demonstrated that the receptor gene is on mouse chromosome 17 and is maternally imprinted. In this paper, we report the cloning and characterization of the mouse IGF-II/MPR gene. The gene is 93 kb long, is composed of 48 exons, and codes for a predicted protein of 2482 amino acids. The extracellular part of the receptor is encoded by exons 1-46, with each of 15 related repeating motifs being determined by parts of 3-5 exons. A single fibronectin type II-like element is found in exon 39. The transmembrane portion of the receptor also is encoded by exon 46, and the cytoplasmic region by exons 46-48. The positions of exon-intron splice junctions are conserved between several of the repeats in the IGF-II/MPR and the homologous extracellular region of the gene for the other known lysosomal sorting receptor, the cation-dependent mannose 6-phosphate receptor. The gene duplications that gave rise to the modern IGF-II/MPR probably occurred before the divergence of mammals, since there is more extensive protein sequence conservation between receptors from different species than between any pair of repeating motifs within a single receptor.

Insulin-Like Growth Factor II (IGF-II) and the IGF-II/ Mannose-6-Phosphate Receptor: The Myth Continues

Insulin-like growth factor II (IGF-II) is a polypeptide hormone with structural and functional homology with IGF-I and pro-insulin. It is now thought that IGF-II acts as a growth factor during fetal life and development. In rats, IGF-II levels in the circulation are high in the fetus and decline rapidly after birth. IGF-II mRNA expression in many tissues including the liver and the choroid plexus is also high during fetal life and low thereafter. Targeted disruption of the IGF-II gene in mice leads to a deficiency in their growth. Alternatively, it has been proposed that IGF-II could act as a growth and differentiation factor in the central nervous system. Administration of IGF-II into the central nervous system in rats leads to increased food intake and altered feeding behaviour. In muscle cells and in colon epithelial cells, IGF-II might also serve as an important regulator of differentiation. A key role for IGF-II as a paracrine or autocrine growth factor in certain tumours has been proposed. The IGFs exert their effects by binding to high-affinity membrane receptors that are expressed in many cells and tissues. The IGF-I receptor, which binds IGF-I with the highest affinity and which is very similar to the insulin receptor, is thought to mediate most, if not all, of the IGF-induced biological functions. The IGF-II/mannose-6-phosphate (M6P) receptor is a bifunctional glycoprotein with no homology to the insulin receptor. This receptor binds IGF-II and lysosomal enzymes bearing the M6P recognition marker at distinct binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Mouse Sertoli cells secrete mannose 6-phosphate containing glycoproteins that are endocytosed by spermatogenic cells.

Sertoli cells were isolated from prepubertal mice and cultured in serum-free medium to determine whether they secrete glycoproteins containing mannose 6-phosphate (M6P). Assays of the conditioned medium for lysosomal enzyme precursors, which typically bear the M6P recognition marker, indicated that Sertoli cells selectively secreted beta-N-acetylhexosaminidase and alpha-mannosidase, but not beta-glucuronidase or beta-galactosidase. Sertoli cells were labeled metabolically with [35S]methionine and the conditioned medium was fractionated on a cation-independent M6P receptor affinity column. Most of the secreted proteins did not bind to the column (peak A); however, approximately 10% of the radioactivity eluted as a low-affinity fraction (peak B), and 5-11% of the recovered cpm bound to the column and were eluted with 2.5 mM M6P (peak C). The radiolabeled proteins in each fraction were analyzed by one- and two-dimensional electrophoresis and fluorography. Two protein bands with molecular weights of 30,000 and 35,000 were present in peak B. Peak C contained at least ten M6P-containing glycoproteins with molecular weights between 30,000 and 135,000 and isoelectric points < 6.5. The 35,000-molecular-weight constituent prominent both in peaks B and C was identified as procathepsin L by immunoprecipitation with a specific antibody. When pachytene spermatocytes and round spermatids were cultured overnight in the presence of peak C glycoproteins radiolabeled with 125I, both germ cell types accumulated these Sertoli M6P-glycoproteins by a receptor-mediated process that was specifically inhibited by M6P. The Sertoli M6P-glycoproteins taken up by germ cells were processed to lower molecular weight forms. These results provide evidence that M6P receptors on the surface of spermatogenic cells endocytose secrete glycoproteins that are likely to be present in the seminiferous epithelium.

Human lysosomal protective protein. Glycosylation, intracellular transport, and association with beta-galactosidase in the endoplasmic reticulum.

In lysosomes beta-galactosidase and neuraminidase acquire a stable and active conformation through their association with the protective protein. The latter is homologous to serine carboxypeptidases and has cathepsin A-like activity which is distinct from its protective function towards the two glycosidases. To define signals in the human protective protein important for its intracellular transport, and to determine the site of its association with beta-galactosidase, we have generated a set of mutated protective protein cDNAs carrying targeted base substitutions. These mutants were either singly transfected into COS-1 cells or cotransfected together with wild type human beta-galactosidase. We show that all point mutations cause either a complete or partial retention of the protective protein precursor in the endoplasmic reticulum. This abnormal accumulation leads to degradation of the mutant proteins probably in this compartment. Only the oligosaccharide chain on the 32-kDa subunit acquires the mannose 6-phosphate recognition marker, the one on the 20-kDa subunit seems to be merely essential for the stability of the mature protein. In cotransfection experiments, wild type beta-galactosidase and protective protein appear to assemble already as precursors, soon after synthesis, in the endoplasmic reticulum. Mutated protective protein precursors that are retained in the endoplasmic reticulum or pre-Golgi complex interact with and withhold normal beta-galactosidase molecules in the same compartments, thereby preventing their normal routing.

Characterization of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase from Acanthamoeba castellanii.

The kinetic properties of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) partially purified from the soil amoeba Acanthamoeba castellanii have been studied. The transferase phosphorylated the lysosomal enzymes uteroferrin and cathepsin D 3-90-fold better than nonlysosomal glycoproteins and 16-83-fold better than a Man9GlcNAc oligosaccharide. Deglycosylated uteroferrin was a potent competitive inhibitor of the phosphorylation of intact uteroferrin (Ki of 48 microM) but did not inhibit the phosphorylation of RNase B or the simple sugar alpha-methylmannoside. Deglycosylated RNase (RNase A) did not inhibit the phosphorylation of RNase B or uteroferrin. These results indicate that purified amoeba GlcNAc-phosphotransferase recognizes a protein domain present on lysosomal enzymes but absent in most nonlysosomal glycoproteins. The transferase also exhibited a marked preference for oligosaccharides containing mannose alpha 1,2-mannose sequences, but this cannot account for the high affinity binding to lysosomal enzymes. A. castellanii extracts do not contain detectable levels of N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase, the second enzyme in the biosynthetic pathway for the mannose 6-phosphate recognition marker. We conclude that A. castellanii does not utilize the phosphomannosyl sorting pathway despite expression of very high levels of GlcNAc-phosphotransferase.

Biosynthesis, processing, and secretion of α-L-fucosidase in lymphoid cells from patients with I-cell disease and pseudo-Hurler polydystrophy

N-Acetylglucosamine 1-phosphotransferase is a key enzyme required for synthesis of the mannose 6-phosphate recognition marker that is used by many newly made acid hydrolases for their transport to lysosomes. It has previously been found that lymphoid cells from patients with I-cell disease and pseudo-Hurler polydystrophy have nearly normal intracellular and intralysosomal activities of several lysosomal acid hydrolases, despite a deficiency of N-acetylglucosamine 1-phosphotransferase. These results suggest that lymphoid cells may provide an important system to investigate alternate mechanisms for targeting newly made acid hydrolases to lysosomes. In the present study, the biosynthesis, processing and secretion of alpha-L-fucosidase in I-cell and pseudo-Hurler lymphoid cells was used as a model system to study the existence of such mechanisms. The level of intracellular alpha-L-fucosidase protein in exponentially growing I-cell or pseudo-Hurler lymphoid cultures was statistically indistinguishable from the mean of 19 control cultures. A 1.5 h [35S]methionine pulse experiment showed that alpha-L-fucosidase is initially synthesized by I-cell, pseudo-Hurler and control cultures as an intracellular form (Mr = 58,000). Companion cultures chased with methionine from 2 to 21 h processed the enzyme to an intracellular form (Mr = 60,000) and an extracellular form (Mr = 62,000). All enzyme forms were glycoproteins with polypeptide chains of Mr 52,000. In control cells incubated with radioactive inorganic phosphate (32Pi), less than 1% of the 32Pi incorporated into alpha-L-fucosidase was associated with carbohydrate chains and greater than 99% with polypeptide chains. In I-cell disease lymphoid cells, the 32Pi incorporated into alpha-L-fucosidase was associated solely with polypeptide chains. A qualitative analysis of phosphorylated residues identified phosphoserine in alpha-L-fucosidase from control and I-cell lymphoid cells. Only alpha-L-fucosidase from control cells contained mannose 6-phosphate. These results are consistent with the proposal that I-cell lymphoid cells may use a mannose 6-phosphate-independent mechanism for routing alpha-L-fucosidase. Additional metabolic labelling experiments demonstrated the presence of 32P-labelled alpha-L-fucosidase in both cells and medium of a control lymphoid culture, but only in cells of an I-cell lymphoid culture. In contrast, alpha-L-fucosidase labelled with [35S]methionine was found in cells and medium of control and I-cell lymphoid cultures. Since phosphoserine was only found to occur in intracellular, but not in extracellular alpha-L-fucosidase of the I-cell culture, we speculate that phosphoserine may be involved in intracellular retention of alpha-L-fucosidase in I-cell lymphoid cells.

Synthesis of a human lysosomal enzyme, β-hexosaminidase B, using the baculovirus expression system

Human lysosomal,β-hexosaminidase exists in two major forms: the A isoform is composed of both α and β chains, while the B form is a homopolymer of β chains. Deficiency of β-hexosaminidase underlies the GM2 gangliosidoses. We have produced active β-hexosamini-dase B in cultured insect (Sf9) cells by isolation of a recombinant insect virus (baculovirus) containing the cDNA for the β chain within the viral polyhedron gene and infection of Sf9 cells with this construct. That portion of the enzyme secreted into the medium, 50%, was purified with concanavalin A Sepharose and subsequent affinity chromatography to yield β-hexosamini-dase B that is 75% pure. The product has an N-terminal amino acid sequence, specific activity, and size ( M r 62,000) similar to that of the enzyme present in cultured human fibroblasts. However, endo H sensitivity studies revealed that the oligosaccharide structures present on recombinant β-hexosaminidase B differ from those found on the enzyme synthesized in the human system. In addition, these structures lack the mannose 6-phosphate recognition marker that targets degradative hydrolases to lysosomes. Despite these differences, recombinant β-hexosaminidase B does serve as a specific substrate for the mannose phosphorylating enzyme, N-acetylglucosaminyl phosphotransferase. Furthermore, the oligosaccharide moieties phosphorylated in vitro match those phosphorylated in vivo, pointing to the conformational integrity of the recombinant enzyme. Generous amounts of easily obtained, easily purified, and properly folded β-hexosaminidase B will facilitate physical structural analysis of the enzyme.

Rat Heart Perfusion as Model System for Enzyme Replacement Therapy in Glycogenosis Type II

Cardiac failure and skeletal muscle weakness are the main clinical features of glycogenosis type II, a lysosomal storage disorder caused by acid alpha-glucosidase deficiency. In our study, we have investigated in a rat heart perfusion-recirculation system whether acid alpha-glucosidase can be taken up from the vascular system into cardiomyocytes. When rat hearts were perfused with mannose 6-phosphate-containing acid alpha-glucosidase purified from bovine testis, a 3- to 4-fold increase of enzyme activity was obtained. Perfusion with human placental acid alpha-glucosidase not containing the mannose 6-phosphate recognition marker did not have such an effect. The presence of bovine testis acid alpha-glucosidase in heart tissue was demonstrated by immunoblotting. Immunocytochemistry provides evidence for uptake of the exogenous enzyme in lysosomes of the cardiomyocytes. The relevance of these findings for enzyme therapy in glycogenosis type II is discussed.

Surface distribution of the mannose 6-phosphate receptors in epithelial Madin-Darby canine kidney cells.

We have analyzed the surface polarity of both the cation-independent (CI-MPR) and the cation-dependent (CD-MPR) mannose 6-phosphate receptors in the epithelial Madin-Darby canine kidney (MDCK) cell line grown on polycarbonate filters. The surface localization was studied by plasma membrane domain-specific surface labeling methods and by confocal microscopy using MPR-specific antibodies. The CI-MPR was shown to be exclusively present on the basolateral cell surface. In contrast, the CD-MPR was expressed neither apically nor basolaterally. However, an intracellular pool of CD-MPR could be detected. In MDCKII-RCAr cells, cell surface CI-MPR was shown to recycle between the basolateral plasma membrane and the trans-Golgi network. After exogalactosylation, cell surface CI-MPR acquired sialic acid residues in a time-dependent manner. Furthermore, the basolateral CI-MPR was shown to be functional. Lysosomal enzymes, bearing the mannose 6-phosphate recognition marker, were taken up from the basolateral medium and endocytosed into the cells. Uptake of lysosomal enzymes from the apical side was insignificant and not MPR mediated. These results extend previous immunoelectron microscopic studies on the intracellular polarity of the CI-MPR (Parton, R. G., Prydz, K., Bomsel, M., Simons, K., and Griffiths, G. (1989) J. Cell Biol. 109, 3259-3272) which showed that the CI-MPR was present in basolateral early endosomes and in late endosomes but absent from apical early endosomes.

Acidification-dependent dissociation of endocytosed insulin precedes that of endocytosed proteins bearing the mannose 6-phosphate recognition marker.

A key step in the sorting of endocytosed ligands from their receptors is dissociation, which is triggered by the acidic pH of endosomes. To determine whether dissociation occurs synchronously for all ligands, we compared in Chinese hamster ovary cells the intracellular dissociation of insulin, which dissociates between pH 6.3 and 7.0, with that of lysosomal hydrolases bearing the mannose 6-phosphate recognition marker (Man-6-P proteins), which dissociate around pH 5.8. Chinese hamster ovary cells were pulsed for 2 min with 125I-insulin, acid-washed to remove surface binding, and chased. During a 40-min period, about 50% of the internalized 125I-insulin was released intact via a retrocytotic pathway. Retrocytosis was not inhibited by monensin, suggesting that the release was not dependent on acidic endosomes. The remaining insulin dissociated from its receptor in an acidification-sensitive manner and was eventually degraded. Dissociation was 70% complete within 5 min of internalization. When cells were similarly incubated with 125I-Man-6-P proteins, about 35% of the internalized radioactivity was released during a 1-h chase, reflecting proteolytic maturation of the Man-6-P proteins. Dissociation of Man-6-P proteins was acidification-dependent (i.e. inhibited by monensin), and was 50% complete after about 11 min. The results indicate that acidification-dependent dissociation of ligands does not occur in a single step and suggest that multiple endocytic compartments are involved in receptor/ligand sorting.

Molecular Cloning of the Uteroferrin-Associated Protein, a Major Progesterone-Induced Serpin Secreted by the Porcine Uterus, and the Expression of its mRNA during Pregnancy

The uteroferrin(Uf)-associated basic proteins (UfAP) are a group of three (Mr = 42K, 48K, and 50K) antigenically related, basic glycoproteins secreted by the porcine uterus under the influence of progesterone (P4) which exist as heterodimers (Mr = 80,000) with the iron-binding acid phosphatase, Uf. Several UfAP cDNA clones from a day-60 pregnant pig uterine endometrial cDNA library have been cloned and sequenced. The UfAP mRNA is approximately 1400 bases long and has a single open reading frame of 1251 bases with two start codons at positions 64 and 79 from the 5'-end. UfAP mRNA content of the endometrium increases as pregnancy proceeds, reaching maximum levels around day 70 and then remaining relatively constant in late gestation (days 70 to 110). The pro-form of the UfAP minus signal sequence appears to be 392 amino acids in length and has four potential N-linked glycosylation sites Asn107, Asn197, Asn243, and Asn315. Comparison of the NH2-terminal sequences of the individual UfAP poly-peptides with the amino acid sequence deduced from the cDNA has indicated a series of at least four posttranslational proteolytic processing steps which generate the various molecular forms of the UfAP. The deduced amino acid sequence of UfAP shares considerable identity with several protease inhibitors and hormone-binding proteins that are members of the serpin superfamily of proteins. The UfAP amino acid sequence also exhibits about 55% sequence identity with the P4-induced uterine milk proteins (UTMP) of the sheep. Since the UfAP and UTMP share many biosynthetic and structural features that include site of biosynthesis in the endometrium, P4-responsiveness, the presence of the mannose 6-phosphate lysosomal recognition marker, and considerable sequence similarity, the UfAP and the UTMP may have homologous function which for both still remains obscure.