Lysosomal hydrolase mannose 6-phosphate uncovering enzyme resides in the trans-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.

The net effects of the Project NetWork return-to-work case management experiment on participant earnings, benefit receipt, and other outcomes.

The Social Security Administration (SSA) initiated Project NetWork in 1991 to test case management as a means of promoting employment among persons with disabilities. The demonstration, which targeted Social Security Disability Insurance (DI) beneficiaries and Supplemental Security Income (SSI) applicants and recipients, offered intensive outreach, work-incentive waivers, and case management/referral services. Participation in Project NetWork was voluntary. Volunteers were randomly assigned to the "treatment" group or the "control" group. Those assigned to the treatment group met individually with a case or referral manager who arranged for rehabilitation and employment services, helped clients develop an individual employment plan, and provided direct employment counseling services. Volunteers assigned to the control group could not receive services from Project NetWork but remained eligible for any employment assistance already available in their communities. For both treatment and control groups, the demonstration waived specific DI and SSI program rules considered to be work disincentives. The experimental impact study thus measures the incremental effects of case and referral management services. The eight demonstration sites were successful in implementing the experimental design roughly as planned. Project NetWork staff were able to recruit large numbers of participants and to provide rehabilitation and employment services on a substantial scale. Most of the sites easily reached their enrollment targets and were able to attract volunteers with demographic characteristics similar to those of the entire SSI and DI caseload and a broad range of moderate and severe disabilities. However, by many measures, volunteers were generally more "work-ready" than project eligible in the demonstration areas who did not volunteer to receive NetWork services. Project NetWork case management increased average annual earnings by $220 per year over the first 2 years following random assignment. This statistically significant impact, an approximate 11-percent increase in earnings, is based on administrative data on earnings. For about 70 percent of sample members, a third year of followup data was available. For this limited sample, the estimated effect of Project NetWork on annual earnings declined to roughly zero in the third followup year. The findings suggest that the increase in earnings may have been short-lived and may have disappeared by the time Project NetWork services ended. Project NetWork did not reduce reliance on SSI or DI benefits by statistically significant amounts over the 30-42 month followup period. The services provided by Project NetWork thus did not reduce overall SSI and DI caseloads or benefits by substantial amounts, especially given that only about 5 percent of the eligible caseload volunteered to participate in Project NetWork. Project NetWork produced modest net benefits to persons with disabilities and net costs to taxpayers. Persons with disabilities gained mainly because the increases in their earnings easily outweighed the small (if any) reduction in average SSI and DI benefits. For SSA and the federal government as a whole, the costs of Project NetWork were not sufficiently offset by increases in tax receipts resulting from increased earnings or reductions in average SSI and DI benefits. The modest net benefits of Project NetWork to persons with disabilities are encouraging. How such benefits of an experimental intervention should be weighed against costs of taxpayers depends on value judgments of policymakers. Because different case management projects involve different kinds of services, these results cannot be directly generalized to other case management interventions. They are nevertheless instructive for planning new initiatives. Combining case and referral management services with various other interventions, such as longer term financial support for work or altered provider incentives, could produc

The development of the Project NetWork administrative records database for policy evaluation.

This article describes the development of SSA's administrative records database for the Project NetWork return-to-work experiment targeting persons with disabilities. The article is part of a series of papers on the evaluation of the Project NetWork demonstration. In addition to 8,248 Project NetWork participants randomly assigned to receive case management services and a control group, the simulation identified 138,613 eligible nonparticipants in the demonstration areas. The output data files contain detailed monthly information on Supplemental Security Income (SSI) and Disability Insurance (DI) benefits, annual earnings, and a set of demographic and diagnostic variables. The data allow for the measurement of net outcomes and the analysis of factors affecting participation. The results suggest that it is feasible to simulate complex eligibility rules using administrative records, and create a clean and edited data file for a comprehensive and credible evaluation. The study shows that it is feasible to use administrative records data for selecting control or comparison groups in future demonstration evaluations.

Molecular cloning and functional expression of two splice forms of human N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase.

We have isolated and sequenced human cDNA and mouse genomic DNA clones encoding N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase (phosphodiester alpha-GlcNAcase) which catalyzes the second step in the synthesis of the mannose 6-phosphate recognition signal on lysosomal enzymes. The gene is organized into 10 exons. The protein sequence encoded by the clones shows 80% identity between human and mouse phosphodiester alpha-GlcNAcase and no homology to other known proteins. It predicts a type I membrane-spanning glycoprotein of 514 amino acids containing a 24-amino acid signal sequence, a luminal domain of 422 residues with six potential N-linked glycosylation sites, a single 27-residue transmembrane region, and a 41-residue cytoplasmic tail that contains both a tyrosine-based and an NPF internalization motif. Human brain expressed sequence tags lack a 102-base pair region present in human liver cDNA that corresponds to exon 8 in the genomic DNA and probably arises via alternative splicing. COS cells transfected with the human cDNA expressed 50-100-fold increases in phosphodiester alpha-GlcNAcase activity proving that the cDNA encodes the subunits of the tetrameric enzyme. Transfection with cDNA lacking the 102-base pair region also gave active enzyme. The complete genomic sequence of human phosphodiester alpha-GlcNAcase was recently deposited in the data base. It showed that our cDNA clone was missing only the 5'-untranslated region and initiator methionine and revealed that the human genomic DNA has the same exon organization as the mouse gene.

[Ambulatory geriatric assessment of 2116 in poor elderly]. / Valoración geriátrica ambulatoria de 2116 adultos mayores pobres.

BACKGROUND: Geriatric assessment quantifies medical, functional, mental and social capabilities and alterations of elders and is the first step to initiate specific intervention programs. AIM: To report the initial geriatric assessment of a program aimed to help poor elders living in Metropolitan Santiago. SUBJECTS AND METHODS: Two thousand one hundred sixteen free living subjects aged 65 to 99 years old (711 males) were subjected to an assessment using a simple geriatric score validated abroad and used previously in Chile. The resulting score ranges from 0 (better) to 5 (worst). RESULTS: Eighty eight percent of elders did not have problems in the functional evaluation. Subjects over 75 years old needed occasional support for the daily activities with higher frequency than younger subjects (12 and 5.4% respectively, p < 0.001) and had a higher frequency of major functional limitations (7.8 and 3.2% respectively, p < 0.001). Mental assessment was considered normal in 89.4% of subjects. Those over 75 years old had a higher frequency of memory disturbances (11.4 and 6.5% respectively) and cognitive alterations (4.6 and 1.8% respectively). Indefinite social support could be received by 84% of subjects, but 7.4% did not have access to this resource. CONCLUSIONS: Geriatric assessment of poor elders gives useful information to identify those subjects that require community help.

Purification and multimeric structure of bovine N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase.

N-Acetylglucosamine-1-phosphodiester alpha-N-Acetylglucosaminidase (EC 3.1.4.45; phosphodiester alpha-GlcNAcase) catalyzes the second step in the synthesis of the mannose 6-phosphate determinant required for efficient intracellular targeting of newly synthesized lysosomal hydrolases to the lysosome. A partially purified preparation of phosphodiester alpha-GlcNAcase from bovine pancreas was used to generate a panel of murine monoclonal antibodies. The anti-phosphodiester alpha-GlcNAcase monoclonal antibody UC1 was coupled to a solid support and used to immunopurify the bovine liver enzyme 670,000-fold in two steps to apparent homogeneity with an overall yield of 14%. The purified phosphodiester alpha-GlcNAcase has a specific activity of 498 micromol of [3H]GlcNAc-alpha-phosphomannose-alpha-methyl cleaved per h per mg of protein using 0.5 mM [3H]GlcNAc-alpha-phosphomannose-alpha-methyl as substrate. The subunit structure of the enzyme was determined using a combination of analytical gel filtration chromatography, SDS-polyacrylamide gel electrophoresis, and amino-terminal sequencing. The data indicate that bovine phosphodiester alpha-GlcNAcase is a 272,000-Da complex of four identical 68,000-Da glycoprotein subunits arranged as two disulfide-linked homodimers. A soluble form of the enzyme, isolated from fetal bovine serum, showed the same subunit structure. Both forms of the enzyme reacted with a rabbit antibody raised to the amino-terminal peptide of the liver enzyme, suggesting that phosphodiester alpha-GlcNAcase is a type I membrane-spanning glycoprotein with its amino terminus in the lumen of the Golgi apparatus.

[Functional risk assessment of poor Chilean elders using an instrument validated in Canada]. / Medición del riesgo funcional en adultos mayores pobres, con un instrumento validado en Canadá.

BACKGROUND: Information about medical and social situation of elders is limited in Chile. AIM: To assess the functional risk of Chilean elders using an instrument validated in Canada. SUBJECTS AND METHODS: As part of a project aimed to help poor elders, 2,116 subjects living in Santiago, aged 65 to 99 years old (1,334 female, and 625 older than 75 years old), were interviewed. RESULTS: Thirty percent of these elders were using more than three medications and 13% lived alone. Visual problems were detected in 75%, memory problems were found in 62%, 63% felt depressed, 46% had hearing problems, 42% suffered a fall during the last year, 35% had a health problem that forced them to stay at home, 32% did not count with help in a case of need, 33% referred some type of nutritional problem, 26% needed help for daily living activities and 25% considered to have a worst health than counterparts of the same age. Among subjects older than 75 years old, the frequency of memory problems, auditory impairment, number of falls, health problems that precluded leaving the house, limitation for daily activities and the use of walking aids, was significantly higher. Although men and women had similar ages, men were in worst functional conditions, and had required more admissions to hospitals. There was a higher proportion of women living alone. Females also had a higher frequency of depression, memory disturbances, falls and use of more than three medications. CONCLUSIONS: Women elders tend to be in better functional conditions than men and people older than 75 years old have a higher functional risk. The applied instrument allowed a better focalization of our geriatric program.

Purification and kinetic parameters of bovine liver N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase.

The enzyme N-acetylglucosamine phosphodiester alpha-N-acetylglucosaminidase (phosphodiester alpha-GlcNAcase) catalyzes the second step in the formation of the mannose 6-phosphate targeting signal on lysosomal enzyme oligosaccharides by removing GlcNAc residues from GlcNAc-alpha-P-mannose moieties, which are formed in the first step by UDP-N-acetyl-glucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase). Phosphodiester alpha-GlcNAcase, a membrane-bound enzyme, has been purified about 3,000-fold from bovine liver to apparent homogeneity using detergent solubilization, fractionation on DEAE-cellulose, affinity chromatography on lectin-Sepharose columns, gel filtration, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme migrated as 129- and 121-kDa species on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since both bands had the same amino-terminal sequence, the smaller species is presumed to be derived from the larger by proteolysis. Kinetic analysis of bovine phosphodiester alpha-GlcNAcase with enzymatically synthesized artificial and biological substrates indicates that phosphodiester alpha-GlcNAcase requires GlcNAc-alpha-P R for substrate and that when R contains the Man alpha 1,2Man linkage the substrate binding is most effective. Unlike GlcNAc-phosphotransferase, bovine phosphodiester alpha-GlcNAcase does not require a protein recognition determinant on lysosomal enzyme substrates.

Characterization and immunolocalization of bovine N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase.

N-Acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase (phosphodiester alpha-GlcNAcase) has been purified 3,000-fold from bovine liver and its kinetic properties determined as described in the previous report (Mullis, K. G., Huynh, M., and Kornfeld, R. (1993) J. Biol. Chem. 269, 1718-1726). This report describes the hydrodynamic and lectin binding properties of phosphodiester alpha-GlcNAcase as well as its intracellular localization. The molecular weight of phosphodiester alpha-GlcNAcase is 204,950, as determined from density gradient centrifugation in D2O and H2O glycerol gradients and gel filtration. Enzymatically active enzyme migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 129,000, consistent with native phosphodiester alpha-GlcNAcase being a dimer. The lectin binding properties of phosphodiester alpha-GlcNAcase indicate that it contains sialylated species of both complex type N-linked oligosaccharides and O-linked oligosaccharides. In immunofluorescence studies phosphodiester alpha-GlcNAcase shows a perinuclear, Golgi localization in Vero cells as does the mid-Golgi marker alpha-mannosidase II. After exposure of the Vero cells to brefeldin A, phosphodiester alpha-GlcNAcase assumes an endoplasmic reticulum staining pattern. In contrast, in cells costained with the trans-Golgi marker wheat germ agglutinin, the wheat germ agglutinin marker assumed an endosomal network appearance after exposure to brefeldin A. These findings indicate that phosphodiester alpha-GlcNAcase is normally located within the Golgi stack, separate from the trans-Golgi and trans-Golgi network stained by wheat germ agglutinin.

Developmental regulation of asparagine-linked oligosaccharide synthesis in Dictyostelium discoideum.

In the preceding report we demonstrated that the expression of two developmentally regulated alpha-mannosidase activities is induced in Dictyostelium discoideum during its differentiation from single-cell amoebae to multicellular organism (Sharkey, D. J., and Kornfeld, R. (1991) J. Biol. Chem. 266, 18477-18484). These activities, designated membrane alpha-mannosidase I (MI) and membrane alpha-mannosidase II (MII), were shown to have several properties in common with rat liver Golgi alpha-mannosidases I and II, respectively, suggesting that MI and MII may play a role in the processing of asparagine-linked oligosaccharides in developing D. discoideum. In this study we analyzed the structures of the asparagine-linked oligosaccharides synthesized by D. discoideum at various stages of development to determine the timing and extent of asparagine-linked oligosaccharide processing. Cells were labeled with [2-3H] mannose, and then total cellular glycoproteins were digested with Pronase to generate glycopeptides that were fractionated on concanavalin A-Sepharose. Glycopeptides from each fraction were digested with endoglycosidase H, both before and after desulfation by solvolysis, and the released, neutral oligosaccharides were sized by high pressure liquid chromatography. At early stages of development, D. discoideum contain predominantly large high mannose-type oligosaccharides (Man9GlcNAc and Man8GlcNAc). Some of these are modified by GlcNAc residues attached beta 1-4 to the mannose-linked alpha 1-6 to the beta-linked core mannose (the "intersecting" position), as well as by fucose, sulfate, and phosphate. In contrast, the oligosaccharides found at late stages of development (18-24 h) have an array of sizes from Man9GlcNAc to Man3GlcNAc. These are still modified by GlcNAc, fucose, sulfate, and phosphate, but the percent of larger high mannose oligosaccharides that are modified with GlcNAc in the intersecting position decreases after 6 h of development, in parallel with the decrease in the intersecting GlcNAc transferase activity. Similarly, the changes in the size of asparagine-linked oligosaccharides synthesized during development correlate well with the appearance of MI and MII activities and suggest that these developmentally regulated alpha-mannosidase activities function in the processing of these oligosaccharides. This is supported further by the observation that oligosaccharide processing was inhibited in late stage cells labeled in the presence of either deoxymannojirimycin, an inhibitor of MI, or swainsonine, an inhibitor of MII.

Developmental regulation of processing alpha-mannosidases and "intersecting" N-acetylglucosaminyltransferase in Dictyostelium discoideum.

We have identified three developmentally regulated oligosaccharide-processing enzyme activities in Dictyostelium discoideum. Two different alpha-mannosidase activities present at extremely low levels in vegetative cells are expressed during development. The first of these activities (MI) rises sharply from 6 to 12 h of development whereas the second activity (MII) rises sharply from 12 to 18 h of development. MI acts on Man9GlcNAc, which it can degrade to Man5GlcNAc but is inactive toward p-nitrophenyl-alpha-D-mannoside (pnpMan). MII acts on pnpMan but not Man9GlcNAc. These activities are distinct from each other and from lysosomal alpha-mannosidase activity as demonstrated by pH optima, substrate specificity, sensitivity to inhibitors and divalent cations, developmental profiles, and solubility. The characteristics of these developmentally regulated alpha-mannosidase activities are similar to those of Golgi alpha-mannosidases I and II from higher eucaryotes, and they appear to catalyze the in vivo formation of processed asparagine-linked oligosaccharides by developed cells. In addition, developed cells have very low levels of a soluble alpha-mannosidase activity, which is the predominant activity in vegetative cells. This soluble vegetative alpha-mannosidase activity has properties that are reminiscent of the endoplasmic reticulum alpha-mannosidase from rat liver. The intersecting N-acetylglucosaminyltransferase activity that we have described recently in vegetative cells of D. discoideum (Sharkey, D. J., and Kornfeld, R. (1989) J. Biol. Chem. 264, 10411-10419) has a developmental profile that is distinct from that of either of the alpha-mannosidase activities. It has maximum activity at 6 h of development and decreases sharply to its minimum level by 12 h of development. The changes that occur in the levels of these three processing enzymes with development correlate well with the different arrays of asparagine-linked oligosaccharides found in early and late stages of development (Sharkey, D. J., and Kornfeld, R. (1991) J. Biol. Chem. 266, 18485-18497).

alpha-Glucosidase II-deficient cells use endo alpha-mannosidase as a bypass route for N-linked oligosaccharide processing.

The kinetics of N-linked oligosaccharide processing and the structures of the processing intermediates have been examined in normal parental BW5147 mouse lymphoma cells and the alpha-glucosidase II-deficient PHAR2.7 mutant cells. The mutant cells accumulated glucosylated intermediates but were able to deglucosylate and process about 40% of their oligosaccharides to complex-type. This processing was not due to residual alpha-glucosidase II activity since the alpha-glucosidase inhibitors 1-deoxynojirimycin (DNJ) and N-butyl-DNJ did not prevent it. Parent cells also showed alpha-glucosidase II-independent processing in the presence of DNJ and N-butyl-DNJ. Membrane preparations from both parent and mutant cells had endo alpha-mannosidase activity, that is, split Glc1,2Man9GlcNAc to Glc1,2Man plus Man8GlcNAc, indicating that this was a candidate for an alternate route to complex oligosaccharide formation in the mutant cells. A balance study in which the cellular glycoproteins, intracellular water soluble saccharides, and saccharides secreted into the medium were isolated and analyzed from [2-3H]mannose-labeled mutant cells showed that the cells formed the di- and trisaccharides Glc1Man and Glc2Man in amounts equivalent to the deglucosylated oligosaccharides found in the cellular glycoproteins. This result shows unequivocally that the alpha-glucosidase II-deficient mutant cells use endo alpha-mannosidase as a bypass route for N-linked oligosaccharide processing.

Identification of an N-acetylglucosaminyltransferase in Dictyostelium discoideum that transfers an "intersecting" N-acetylglucosamine residue to high mannose oligosaccharides.

Glycoproteins synthesized by the cellular slime mold Dictyostelium discoideum have been shown to contain asparagine-linked high-mannose oligosaccharides which have an N-acetylglucosamine group in a novel intersecting position (attached beta 1-4 to the mannose linked alpha 1-6 to the core mannose). We have used crude membrane preparations from vegetative D. discoideum (strain M4) to characterize the enzyme activity responsible for catalyzing the transfer of GlcNAc to the intersecting position of high-mannose oligosaccharides. UDP-GlcNAc:oligosaccharide beta-N-acetylglucosaminyltransferase activity in these preparations attaches GlcNAc to the mannose residue-linked alpha 1-6 to the beta-linked core mannose of the following Man9GlcNAc oligosaccharide as shown by the arrow. (formula; see text) It will also attach GlcNAc to the same intersecting position and/or to the bisecting position (beta-linked core mannose) of the following Man5GlcNAc oligosaccharide. (formula; see text) An analysis of the pH profiles, effects of heat denaturation, and substrate inhibitions on the addition of GlcNAc to either the intersecting or bisecting position of this Man5GlcNAc oligosaccharide indicates that a single enzyme activity is responsible for transferring GlcNAc to both positions. Various oligosaccharides were assayed to determine the substrate specificity of the transferase activity. These data indicate that both the mannose-attached alpha 1-3 and the mannose-attached alpha 1-6 to the mannose receiving the GlcNAc play a critical role in substrate suitability; absence of the alpha 1-6 mannose results in at least a 90% decrease in activity, while absence of the alpha 1-3 mannose results in a completely inactive substrate. This suggests that the minimal substrate is the disaccharide Man alpha 1-3Man.

Post-translational protein modification in the endoplasmic reticulum. Demonstration of fatty acylase and deoxymannojirimycin-sensitive alpha-mannosidase activities.

We have previously described a hybrid protein, GHHA, that contains a fragment of the influenza hemagglutinin joined to the C terminus of a nearly complete rat growth hormone (Rizzolo, L.J., Finidori, J., Gonzalez, A., Arpin, M., Ivanov, I.E., Adesnik, M., and Sabatini, D.D. (1985) J. Cell Biol. 101, 1351-1362). GHHA was transported from the rough endoplasmic reticulum (ER) to a smooth cisterna, continuous with the rough ER, but proximal to the Golgi apparatus. We have now labeled GHHA with [3H]palmitate, demonstrating that fatty acylation can occur in the ER. As expected for a thioester linkage, the label was released from GHHA by hydroxylamine and identified as palmitic acid by thin-layer chromatography. In a second study, we analyzed the structure of the N-linked carbohydrate chain of GHHA. The N-linked oligosaccharides, all high-mannose type, were released by endoglycosidase H and size-fractionated by high pressure liquid chromatography. The predominant structures were Glc1Man8GlcNAc and Man8GlcNAc, indicating that only 2 or 3 glucose and 1 mannose residues were removed from the original Glc3Man9GlcNAc2. Determination of the structure by acetolysis fragmentation indicated that a single Man8GlcNAc isomer was formed by a deoxymannojirimycin-sensitive alpha-mannosidase. This contrasts with a previously characterized ER alpha-mannosidase (Bischoff, J., Liscum, L., and Kornfeld, R. (1986) J. Biol. Chem. 261, 4766-4774) that generates the same isomer, but is deoxymannojirimycin-resistant. These data suggest the possibility that different enzymes are partitioned within the ER.

The use of 1-deoxymannojirimycin to evaluate the role of various alpha-mannosidases in oligosaccharide processing in intact cells.

The mannose analogue, 1-deoxymannojirimycin, which inhibits Golgi alpha-mannosidase I but not endoplasmic reticulum (ER) alpha-mannosidase has been used to determine the role of the ER alpha-mannosidase in the processing of the asparagine-linked oligosaccharides on glycoproteins in intact cells. In the absence of the inhibitor, the predominant oligosaccharide structures found on the ER glycoprotein 3-hydroxy-3-methylglutaryl-CoA reductase in UT-1 cells are single isomers of Man6GlcNAc and Man8GlcNAc. In the presence of 150 microM 1-deoxymannojirimycin, the Man8GlcNAc2 isomer accumulates indicating that the 1-deoxymannojirimycin-resistant ER alpha-mannosidase is responsible for the conversion of Man9GlcNAc2 to Man8GlcNAc2 on reductase. The processing of Man8GlcNAc2 to Man6GlcNAc2, however, must be attributed to a 1-deoxymannojirimycin-sensitive alpha-mannosidase. When cells were radiolabeled with [2-(3)H]mannose for 15 h in the presence of 1-deoxymannojirimycin and then further incubated for 3 h in nonradioactive medium without inhibitor, the Man8GlcNAc2 oligosaccharides which accumulated during the labeling period were partially trimmed to Man6GlcNAc. This finding suggests that a second alpha-mannosidase, sensitive to 1-deoxymannojirimycin, resides in the crystalloid ER and is responsible for trimming the reductase oligosaccharide chain from Man8GlcNAc2 to Man6GlcNAc2. To determine if ER alpha-mannosidase is responsible for trimming the oligosaccharides of all glycoproteins from Man9GlcNAc to Man8GlcNAc, the total asparagine-linked oligosaccharides of rat hepatocytes labeled with [2-(3)H]mannose in the presence or absence of 1.0 mM 1-deoxymannojirimycin were examined. the inhibitor prevented the formation of complex oligosaccharides and caused a 30-fold increase in the amount of Man9GlcNAc2 and a 13-fold increase in the amount of Man8GlcNAc2 present on secreted glycoproteins. This result suggests that only one-third of the secreted glycoproteins is initially processed by ER alpha-mannosidase, and two-thirds are processed by Golgi alpha-mannosidase I or another 1-deoxymannojirimycin-sensitive alpha-mannosidase. The inhibitor caused only a 2.6-fold increase in the amount of Man9GlcNAc2 on cellular glycoproteins suggesting that a higher proportion of these glycoproteins are initially processed by the ER alpha-mannosidase. We conclude that some, but not all, hepatocyte glycoproteins are substrates for ER alpha-mannosidase which catalyzes the removal of a specific mannose residue from Man9GlcNAc2 to form a single isomer of Man8GlcNAc2.

The soluble form of rat liver alpha-mannosidase is immunologically related to the endoplasmic reticulum membrane alpha-mannosidase.

The soluble alpha-mannosidase of rat liver, originally described as a cytoplasmic alpha-mannosidase, has been purified to homogeneity by conventional techniques. The purified enzyme has an apparent molecular weight of 350,000 and is composed of 107-kDa subunits. The soluble alpha-mannosidase has the same enzymatic properties as the endoplasmic reticulum (ER) membrane alpha-mannosidase of rat liver (Bischoff, J., and Kornfeld, R. (1983) J. Biol. Chem. 258, 7909-7910) which is believed to play a role in oligosaccharide processing in the rough ER. Like the membrane-bound ER alpha-mannosidase, the soluble alpha-mannosidase can hydrolyze alpha-linked mannose from both p-nitrophenyl alpha-mannoside (Km = 0.14 mM) and high mannose oligosaccharides, is not inhibited by the mannose analogues swainsonine and 1-deoxymannojirimycin, is stabilized by MnCl2 or CoCl2, and does not bind to concanavalin A-Sepharose. A goat polyclonal antibody raised against the purified soluble alpha-mannosidase specifically recognizes the rat liver membrane-bound ER alpha-mannosidase, leading us to propose that they are two forms of the same enzyme and that the soluble form is derived from the ER membrane alpha-mannosidase by proteolysis. The antibody also cross-reacts with both the soluble and membrane-bound forms of ER alpha-mannosidase activity in cultured Chinese hamster ovary cells and rat H35 hepatoma cells. Since the ER alpha-mannosidase is presumed to be involved in the early steps of oligosaccharide processing, the action of the purified soluble form of the enzyme on high mannose oligosaccharides was examined. Surprisingly, the enzyme released free mannose from oligosaccharides ranging in size from Glc1Man9GlcNAc to Man5GlcNAc with almost equal efficiency. However, a long term incubation of the enzyme with Man9GlcNAc led to the accumulation of Man7GlcNAc and produced only small amounts of Man6GlcNAc and Man5GlcNAc. Structural analysis of these reaction products indicated that the purified soluble form of ER alpha-mannosidase shows little specificity for which mannose residues it removes from Man9GlcNAc. In contrast, as shown in the accompanying paper, the intracellular action of ER alpha-mannosidase on glycoprotein-bound Man9GlcNAc2 is highly specific.

The effect of 1-deoxymannojirimycin on rat liver alpha-mannosidases.

The mannose analogue, 1-deoxymannojirimycin, has been tested for its effect on five alpha-mannosidase activities present in rat liver and shown to be a specific inhibitor of Golgi alpha-mannosidase I at low mumolar concentrations. Golgi alpha-mannosidases I and II were assayed in a highly purified Golgi membrane preparation. Endoplasmic reticulum alpha-mannosidase activity was measured in a rough endoplasmic reticulum detergent extract. A purified soluble alpha-mannosidase activity which we believe is derived from the endoplasmic reticulum during tissue homogenization was also tested. And finally, the lysosomal or acidic alpha-mannosidase was measured in a postnuclear supernatant fraction obtained from rat liver. The results presented here show that 1-deoxymannojirimycin inhibits only Golgi alpha-mannosidase I, which is consistent with its effect on oligosaccharide processing in vivo (Fuhrmann et al. Nature 1984 307:755-758).

The antigen identified by a mouse monoclonal antibody raised against human renal cancer cells is the adenosine deaminase binding protein.

The antigen recognized by a mouse monoclonal antibody (mAb S27) raised against a human renal cancer cell line has been identified as the adenosine deaminase binding protein. mAb S27 immunoprecipitates binding protein purified from a soluble fraction of human kidney. It also recognizes the mature 120,000-dalton membrane form of binding protein from [35S]methionine-labeled human fibroblasts, HepG2 cells, and the renal cancer cell line against which the antibody was raised. A rabbit polyclonal antibody raised against purified kidney binding protein completely precipitates mAb S27-reactive material from labeled membrane extracts. mAb S27 does not precipitate the initially synthesized 110,000 molecular weight precursor of binding protein in fibroblasts and recognizes only a small portion of binding protein precursor in labeled HepG2 cells suggesting that the antigenic determinant recognized by mAb S27 may be a post-translational modification present on the mature form of binding protein or that mAb S27 recognizes molecules in a certain conformation. Glycopeptides derived from purified soluble kidney binding protein or exogenously added adenosine deaminase do not inhibit the immunoprecipitation of binding protein by mAb S27, indicating that the mature oligosaccharide chains of binding protein are not the determinant recognized by mAb S27 and that bound adenosine deaminase does not mask the antigenic sites on binding protein. The fact that monoclonal antibody S27, previously shown (Ueda, R., Ogata, S., Morissey, D. M., Finstad, C. L., Szkudlavek, J., Whitmore, W. F., Oettgen, H. F., Lloyd, K. O., and Old, L. J. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 5122-5126) to detect a cell surface antigen on cultured renal cancer cells, is directed against the adenosine deaminase binding protein confirms and extends the earlier observation (Andy, R.J., and Kornfeld, R. (1982) J. Biol. Chem. 257, 7922-7925) that binding protein is located on the cell surface.