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Viruses, despite their simple structural composition, engage in intricate and complex interactions with their hosts due to their parasitic nature. A notable demonstration of viral behavior lies in their exploitation of lysosomes, specialized organelles responsible for the breakdown of biomolecules and clearance of foreign substances, to bolster their own replication. The man-nose-6-phosphate (M6P) pathway, crucial for facilitating the proper transport of hydrolases into lysosomes and promoting lysosome maturation, is frequently exploited for viral manipulation in support of replication. Recently, the discovery of lysosomal enzyme trafficking factor (LYSET) as a pivotal regulator within the lysosomal M6P pathway has introduced a fresh perspective on the intricate interplay between viral entry and host factors. This groundbreaking revelation illuminates unexplored dimensions of these interactions. In this review, we endeavor to provide a thorough overview of the M6P pathway and its intricate interplay with viral factors during infection. By consolidating the current understanding in this field, our objective is to establish a valuable reference for the development of antiviral drugs that selectively target the M6P pathway., Competing Interests: QZ is employed by Shanghai Sino Organoid Lifesciences Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Liu, Wang, Xu, Zhang and Wang.)

Despite the worldwide consumption of bovine milk, dairy products from small ruminants, such as goat's and sheep's milk, are gaining a large interest especially in the Mediterranean area. The aim of this work was to study the metabolite profiles of 30 sheep's and 28 goat's milk using an untargeted metabolomics approach by a gas chromatography coupled with mass spectrometry (GC-MS) analysis. Results showed several differences in the metabolite profiles: arabitol, citric acid, α-ketoglutaric acid, glyceric acid, myo-inositol, and glycine were more abundant in sheep's milk, while goat's milk had higher levels of mannose-6-phosphate, isomaltulose, valine, pyroglutamic acid, leucine, and fucose. Associations between metabolite profile and milk compositional traits were also found. Predictive capabilities of statistical models indicated a good correlation between the metabolite profile and the protein content in sheep's milk, and with the fat content in goat's milk. This work leads to a better understanding of milk metabolites in small ruminants and their role in the evaluation of milk properties., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Mannose-6-phosphate (M-6-P) glycan analysis is important for quality control of therapeutic enzymes for lysosomal storage diseases. Here, we found that the analysis of glycans containing two M-6-Ps was highly affected by the hydrophilicity of the elution solvent used in high-performance liquid chromatography (HPLC). In addition, the performances of three fluorescent tags--2-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB), and 3-(acetyl-amino)-6-aminoacridine (AA-Ac)--were compared with each other for M-6-P glycan analysis using HPLC and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The best performance for analyzing M-6-P glycans was shown by 2-AA labeling in both analyses., (Copyright © 2016 Elsevier Inc. All rights reserved.)

High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) is an established technique for the carbohydrate analysis of glycoproteins. HPAE-PAD is routinely used for determinations of monosaccharide, sialic acid, mannose-6-phosphate (M-6-P), and oligosaccharide contents of a glycoprotein. This is true for both the initial investigation of a glycoprotein and routine assays of recombinant therapeutic glycoproteins. This contribution reviews the fundamentals of HPAE-PAD, recent technological improvements, and advances in the last ten years in its application to carbohydrate analysis of glycoproteins. The application areas reviewed include monosaccharide determinations, sialic acid determinations, M-6-P determinations, sugar alcohol determinations, analysis of polysialic acids, neutral and charged oligosaccharide analysis, following glycosidase and glycosyltransferase reactions, and coupling HPAE-PAD to mass spectrometry (MS).

A novel method was developed for the determination of mannose-6-phosphate and the phosphate in pharmaceutical intermediates by high performance ion-exchange chromatography (IC) and electrochemical detection. The sample was dissolved with purified water, filtrated by a 0.22 microm nylon filter membrane, and then separated by ion-exchange chromatography. The separation was performed on an IonPac AS18 column (250 mm x4 mm) with an IonPac AG18 (50 mm x4 mm) as guard column, and 25 mmol/L KOH solution as the eluent at a flow rate of 1.0 mL/min. The detection was performed with electrochemical detectors (an integrated pulsed amperometric detector and a suppressed conductivity detector in line). Mannose-6-phosphate was selectively determined with an integrated pulsed amperometric detector first. Then, both of mannose-6-phosphate and phosphate were determined with a suppressed conductivity detector after the background conductance of KOH was suppressed with the ASRS suppressor. The injection volume was 25 microL. The external standard calibration curve was used for quantitative analysis. With an amperometric detector, the linear range of the method for mannose-6-phosphate were 0.06 - 10.0 mg/L (r = 0.9998). The recoveries were 92.1% - 103.1% with the relative standard deviations (RSDs) less than 3%. The detection limit was 0.02 mg/L for mannose-6-phosphate. The method is simple, effective, sensitive and selective. It can be used to determine the quality of pharmaceutical intermediates.

Phosphomannomutase (PMM) deficiency causes congenital disorder of glycosylation (CDG)-Ia, a broad spectrum disorder with developmental and neurological abnormalities. PMM converts mannose 6-phosphate (M6P) to mannose-1-phosphate, a precursor of GDP-mannose used to make Glc(3)Man(9)GlcNAc(2)-P-P-dolichol (lipid-linked oligosaccharide; LLO). LLO, in turn, is the donor substrate of oligosaccharyltransferase for protein N-linked glycosylation. Hepatically produced N-linked glycoproteins in CDG-Ia blood are hypoglycosylated. Upon labeling with [(3)H]mannose, CDG-Ia fibroblasts have been widely reported to accumulate [(3)H]LLO intermediates. Since these are thought to be poor oligosaccharyltransferase substrates, LLO intermediate accumulation has been the prevailing explanation for hypoglycosylation in patients. However, this is discordant with sporadic reports of specific glycoproteins (detected with antibodies) from CDG-Ia fibroblasts being fully glycosylated. Here, fluorophore-assisted carbohydrate electrophoresis (FACE, a nonradioactive technique) was used to analyze steady-state LLO compositions in CDG-Ia fibroblasts. FACE revealed that low glucose conditions accounted for previous observations of accumulated [(3)H]LLO intermediates. Additional FACE experiments demonstrated abundant Glc(3)Man(9)GlcNAc(2)-P-P-dolichol, without hypoglycosylation, CDG-Ia fibroblasts grown with physiological glucose. This suggested a "missing link" to explain hypoglycosylation in CDG-Ia patients. Because of the possibility of its accumulation, the effects of M6P on glycosylation were explored in vitro. Surprisingly, M6P was a specific activator for cleavage of Glc(3)Man(9)GlcNAc(2)-P-P-dolichol. This led to futile cycling the LLO pathway, exacerbated by GDP-mannose/PMM deficiency. The possibilities that M6P may accumulate in hepatocytes and that M6P-stimulated LLO cleavage may account for both hypoglycosylation and the clinical failure of dietary mannose therapy with CDG-Ia patients are discussed.

Congenital disorders of glycosylation (CDG) are a group of multisystemic disorders resulting from defects in the synthesis and processing of N-linked oligosaccharides. The most common form, CDG type Ia (CDG-Ia), results from a deficiency of the enzyme phosphomannomutase (PMM). PMM converts mannose 6-phosphate (man-6-P) to mannose-1-phosphate (man-1-P), which is required for the synthesis of GDP-mannose, a substrate for dolichol-linked oligosaccharide synthesis. The traditional assay for PMM, a coupled enzyme system based on the reduction of NADP(+) to NADPH using man-1-P as a substrate, has limitations in accuracy and reproducibility. Therefore, a more sensitive, direct test for PMM activity, based on the detection of the conversion of man-1-P to man-6-P by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), was developed. Using this assay, the activity of PMM was markedly deficient in fibroblasts and lymphoblasts from 23 patients with CDG-Ia (range 0-15.3% of control, average 4.9+/-4.7%) and also decreased in seven obligate heterozygotes (range 33.0-72.0% of control, average 52.2+/-14.7%). Unlike the spectrophotometric method, there was no overlap in PMM activity among patients, obligate heterozygotes, or controls. Thus, the PMM assay based on HPAEC-PAD has increased utility in the clinical setting, and can be used, together with transferrin isoelectric focusing, to diagnose patients with CDG-Ia and to identify heterozygotes when clinically indicated.

Leishmania promastigotes express an abundant cell surface glycoconjugate, lipophosphoglycan (LPG). LPG contains a polymer of the disaccharide-phosphate repeat unit Galbeta1,4Manalpha1-PO4, shared by other developmentally regulated molecules implicated in parasite virulence. Functional complementation of a Leishmania donovani LPG-defective mutant (OB1) accumulating a truncated LPG containing only the Manalpha1-PO4 residue of the first repeat unit identified LPG3, the Leishmania homolog of the mammalian endoplasmic reticulum (ER) chaperone GRP94. LPG3 resembles GRP94, as it localizes to the parasite ER, and lpg3(-) mutants show defects including down-regulation of surface GPI-anchored proteins and mild effects on other glycoconjugates. LPG3 binds cellular proteins and its Leishmania infantum GRP94 ortholog is highly immunogenic, suggesting a potential role in directing the immune response. However, null lpg3(-) mutants grow normally, are completely defective in the synthesis of phosphoglycans, and the LPG3 mRNA is regulated developmentally but not by stress or heat. Thus the role of LPG3/GRP94 in Leishmania metabolism differs significantly from other eukaryotes. Like the other glycoconjugate synthetic pathways in this parasite, its activity is focused on molecules implicated in virulence rather than viability.

An assay has been developed to quantitate the amount of mannose 6-phosphate in glycoproteins using high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The method was tested on a recombinant lysosomal enzyme, human alpha-galactosidase A, that contains mannose 6-phosphate. The assay includes two steps: hydrolysis of the glycoprotein in 6.75 M trifluoroacetic acid to release mannose 6-phosphate and quantitation of the released mannose 6-phosphate using HPAEC with PAD. There is a linear relationship between the amount of mannose 6-phosphate measured and the amount of alpha-galactosidase hydrolyzed. The assay is also sensitive for as little as 2.5 microg alpha-galactosidase, which contains 117 pmol mannose 6-phosphate. Further, the assay has been shown to have good day-to-day and operator-to-operator consistency. In order to evaluate the assay for glycoprotein in crude extract, the glycoprotein was separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane. The amount of mannose 6-phosphate in the electroblots following hydrolysis was determined using HPAEC-PAD. The assay was also linear when measuring mannose 6-phosphate on electroblots. Therefore, this assay has been shown to be specific, sensitive, and reproducible.

In this paper, we illustrate the fine structural localization of distinct marker proteins in the organelles of human neutrophils and outline our preferred methods for processing ultrathin cryosections for use with immunoelectron microscopy. Previous work has determined the subcellular localization of certain marker proteins within intact polymorphonuclear neutrophilic leukocytes (PMN) and PMN fractions. These are as follows: myeloperoxidase (MPO) for azurophilic granules, lactoferrin for specific/secondary granules, gelatinase for gelatinase/tertiary granules, albumin for the secretory vesicles, and HLA class I and L-selectin for the plasma membrane. In addition to analyzing the heterogeneity of the PMN granule populations, new information on the lysosomal system of this cell is reviewed and extended by the localization of the lysosome-associated membrane proteins (LAMPs) and the cation-independent mannose 6-phosphate receptor (CI-M6PR). LAMPs were absent in all identified granule populations, but were found in the membranes of vesicles, multivesicular bodies (MVB), and multilaminar compartments (MLC). We show here that MVB contain CI-M6PR whereas MLC do not. Furthermore, since MLC contain LAMPs but not the receptor, they probably correspond to the late endosome. By current criteria, the true lysosomes of the resting PMN are MVB and MLC. Finally, although azurophil granules contain acid hydrolases their membranes do not contain LAMPs and they cannot be classified as lysosomes, but rather are more similar to regulated secretory granules.

The use of recombinant lysosomal enzymes for enzyme replacement therapy (ERT) is likely to be a necessary component of effective treatment regimens for lysosomal storage diseases (LSDs). The mechanism and rate of uptake into target cells, rate of disappearance of the enzyme from plasma, and its tissue distribution are important factors to assess the need for possible modifications to the enzyme, particularly for LSDs that affect the central nervous system (CNS). Two recombinant lysosomal enzymes, caprine N-acetylglucosamine-6-sulfatase (rc6S) and human N-acetylgalactosamine-4-sulfatase (rh4S), deficient in MPS IIID and MPS VI, respectively, were radiolabeled and purified. The major portion (>77%) of each recombinant enzyme contained the mannose-6-phosphate (M6P) recognition marker as demonstrated by their ability to bind to a M6P receptor affinity column. The uptake of 3H-rc6S and 3H-rh4S into cultured rat brain cells was also inhibited by the addition of 5 mM M6P to the culture medium. After iv administration of 0.4-0.5 mg/kg of 3H-rc6S and 1 mg/kg of 3H-rh4S to the rat, both enzymes were rapidly lost from the circulation in a biphasic fashion (t1/2 for 3H-rc6S = 1.25+/-0.15 min and 37.17+/-23.29 min; t1/2 for 3H-rh4S = 0.41 and 5.3 min). At this dose, about 6% of 3H-rc6S, but only 0.49% of 3H-rh4S, remained in the plasma 4 h after administration, whereas approx 30% of 3H-rc6S and more than 50% of 3H-rh4S was found in the liver. At doses of 1.6-2.0 mg/kg of 3H-rc6S and 1 mg/kg 3H-rh4S, but not at the lower dose of 3H-rc6S, trace levels of both 3H-rc6S and 3H-rh4S were detected in the brain. The low level of enzyme recovered from the brain suggests that modification of rc6S will be necessary to achieve sufficient enzyme uptake into the CNS for effective therapy of MPS IIID.

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, i.e., 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.

Classical late-infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal neurodegenerative disease whose defective gene has remained elusive. A molecular basis for LINCL was determined with an approach applicable to other lysosomal storage diseases. When the mannose 6-phosphate modification of newly synthesized lysosomal enzymes was used as an affinity marker, a single protein was identified that is absent in LINCL. Sequence comparisons suggest that this protein is a pepstatin-insensitive lysosomal peptidase, and a corresponding enzymatic activity was deficient in LINCL autopsy specimens. Mutations in the gene encoding this protein were identified in LINCL patients but not in normal controls.

Many Dictyostelium lysosomal enzymes contain mannose-6-phosphate (Man-6-P) in their N-linked oligosaccharide chains. We have now characterized a new group of lysosomal proteins that contain N-acetylglucosamine-1-phosphate (GlcNAc-1-P) linked to serine residues. GlcNAc-1-P-containing proteins, which include papain-like cysteine proteinases, cofractionate with the lysosomal markers and are in functional vesicles of the endosomal/lysosomal pathway. Immunoblots probed with reagents specific for each carbohydrate modification indicate that the lysosomal proteins are modified either by Man-6-P or GlcNAc-1-P, but not by both. Confocal microscopy shows that the two sets of proteins reside in physically and functionally distinct compartments. Vesicles with GlcNAc-1-P fuse with nascent bacteria-loaded phagosomes less than 3 minutes after ingestion, while those with Man-6-P do not participate in bacterial digestion until about 15 minutes after phagocytosis. Even though both types of vesicles fuse with phagosomes, GlcNAc-1-P- and Man-6-P-bearing proteins rarely colocalize. Since both lysosomal enzymes and their bound carbohydrate modifications are stable in lysosomes, a targeting or retrieval mechanism based on these carbohydrate modifications probably establishes and/or maintains segregation.

Most newly synthesized lysosomal enzymes contain a transient carbohydrate modification, mannose 6-phosphate (Man-6-P), which signals their vesicular transport from the Golgi to the lysosome via Man-6-P receptors (MPRs). We have examined Man-6-P glycoproteins in human urine by using a purified soluble fragment of the soluble cation-independent MPR (sCI-MPR) as a preparative and analytical affinity reagent. In a survey of urine samples from seven healthy subjects, the pattern of Man-6-P glycoproteins detected with iodinated sCI-MPR as a probe in a blotting assay was essentially identical in each, regardless of sex or age. Two bands of approx. 100 and 110 kDa were particularly prominent. Man-6-P glycoproteins in human urine were purified by affinity chromatography on immobilized sCI-MPR. Seven distinct bands revealed by SDS/PAGE and Coomassie Blue staining were subjected to N-terminal sequence analysis. The prominent 100 and 110 kDa Man-6-P glycoproteins were identified as N-acetylglucosamine-6-sulphatase and alpha-glucosidase respectively. This identification was confirmed by molecular mass determinations on the two major bands after deglycosylation. Sequence analysis revealed arylsulphatase A and several previously unidentified proteins as minor species. Man-6-P glycoproteins were also purified on an analytical scale to determine the proportion of a number of lysosomal enzyme activities represented by the mannose-6-phosphorylated forms. The lysosomal enzymes in urine containing the highest proportion of mannose-6-phosphorylated form were beta-mannosidase (82%), hexosaminidase (27%) and alpha-glucosidase (24%). The profiles of Man-6-P glycoproteins detected by blotting in urine and plasma were not similar, suggesting that the urinary species are not derived from the bloodstream.

The reversal line demarcates the cessation of osteoclast activity from the commencement of osteoblast activity at a remodeling site in bone. It is a seam between segments of bone that are formed at different times. We believe that the reversal line contains regulatory signals that, in part, control osteoblast activity. We have conducted a pilot study to examine the fate of reversal lines during abnormal bone remodeling in alveolar bone. A surgical periodontal defect was created in a Cynomolgus monkey (Macaca fascicularis), allowed to heal in the presence of plaque, and evaluated histologically. In this model, there is an acute inflammatory reaction followed by compromised bone formation. Woven bone rather than lamellar bone was deposited in the defect. A striking finding in this wound-healing model was the disruption of the carbohydrate material along the reversal line. This supports our theory that disruption of the signaling molecules in the reversal line may be responsible for uneven woven bone formation.

The formation of a non-covalent complex between mature transforming growth factor beta 1 (TGF-beta 1) and its pro region, the beta 1-latency-associated peptide (beta 1-LAP), is important in regulating the activity of this multipotent growth factor. We have overexpressed simian beta 1-LAP in Chinese hamster ovary (CHO) cells to produce a cell line which secretes beta 1-LAP into the culture medium at > 1 mg/l, thus enabling structural studies of complex formation between beta 1-LAP and TGF-beta 1. The simian beta 1-LAP expressed in CHO cells reversed the growth inhibitory effect of exogenous TGF-beta 1 on Mv1Lu (mink lung epithelial) cells and was able to form a cross-linked complex with 125I-TGF-beta 1. Simian beta 1-LAP was purified to homogeneity by a combination of ammonium sulphate precipitation, gel filtration, dye ligand chromatography and anion-exchange chromatography, with a yield of 15%. The purified protein had an apparent molecular mass of 114 kDa as determined by SDS/PAGE, which is greater than that determined for the transient expression of simian beta 1-LAP in COS-1 and for the simian precursor of TGF-beta 1 (pro-TGF-beta 1) in CHO cells, this major difference being due to more extensive glycosylation of beta 1-LAP expressed by this CHO clone. Far-UV CD spectroscopy of simian beta 1-LAP indicates a mostly beta-sheet structure, with extensive structural rearrangements occurring upon formation of the latent complex between TGF-beta 1 and beta 1-LAP.

Glycolipids synthesized in a cell-free system prepared from the free-living protozoan Paramecium primaurelia and labelled with [3H]mannose and [3H]glucosamine using GDP-[3H]mannose and UDP-[3H]N-acetyl glucosamine, respectively, were identified and structurally characterized as glycosylinositol-phosphoceramides (GIP-ceramides). The ceramide-based lipid was also found in the GIP membrane anchor of the G surface antigen of P.primaurelia, strain 156. Using a combination of in vitro labelling with GDP-[3H]mannose and in vivo labelling with 33P, we found that the core glycans of the P.primaurelia GIP-ceramides were substituted with an acid-labile modification identified as mannosyl phosphate. The modification of the glycosylinositol-phospholipid core glycan by mannosyl phosphate has not been described to date in other organisms. The biosynthesis of GIP-ceramide intermediates in P.primaurelia was studied by a pulse-chase analysis. Their structural characterization is reported. We propose the following structure for the putative GIP-ceramide membrane anchor precursor of P.primaurelia surface proteins: ethanolamine phosphate-6Man-alpha 1-2Man-alpha 1-6Man-(mannosyl phosphate)-alpha 1-4glucosamine-inositol-phosphoceramide.

Newly synthesized enzymes destined for lysosomal localization contain mannose 6-phosphate (Man6-P) residues, allowing interaction with Man6-P receptors (MPRs) and subsequent intracellular targeting to the lysosome. In most cultured cells, lysosomal enzymes are rapidly dephosphorylated after targeting, but in some transformed cell lines, these proteins retain the Man6-P marker. To investigate the significance of this in human malignancy, we examined the persistence of the Man6-P marker in human breast biopsy specimens using MPR derivatives as affinity probes. In one approach, extracts of frozen tissue were standardized to protein content, fractionated by SDS-PAGE, immobilized on nitrocellulose, and probed with iodinated MPR. On average, carcinomas contained 4-fold higher levels of Man6-P glycoproteins than did benign tumors or normal breast samples. In about 15% of the carcinomas, levels of Man6-P glycoproteins were highly elevated (7-10-fold). Multiple Man6-P glycoproteins were detected, suggesting a general alteration in the synthesis or processing of many lysosomal enzymes in carcinomas. In a second approach, sections of formalin-fixed breast biopsy specimens were probed with biotinylated MPR. Malignant cells in 25 of 75 carcinomas exhibited granular cytoplasmic staining in what appears to be intracellular vesicles. Staining was specifically inhibited by Man6-P and was not observed in stromal components or lymphocytes. In addition, Man6-phosphorylated proteins were not detected in the 14 normal or benign biopsy samples examined. Staining appeared to be independent of most prognostic factors examined, including p53, cathepsin D, DNA ploidy, and hormone (estrogen and progesterone) receptor status. However, positive staining was significantly associated with high histological and nuclear grades (P < 0.05) and potentially with c-erbB-2 (P < 0.10), suggesting that elevated levels of Man6-P glycoproteins are associated with the more aggressive tumors.

A sialidase resistant mono-charged N-glycan was isolated from glycosylation site I (Asn-24) of recombinant human erythropoietin expressed from baby hamster kidney (BHK-21) cells and constituted approximately 2-4% of the oligosaccharide material at this glycosylation site. Mass spectrometry and both 1- and 2-dimensional NMR techniques revealed a high mannose type structure (Man6) with a phospho-diesterbridged N-acetylglucosamine as follows: [formula: see text]

Leptospiral lipopolysaccharides (LPS) are the main antigens responsible for immunity in leptospirosis. In this investigation we studied the nature of the antigenic determinants of LPS extracted from Leptospira interrogans serovar hardjo (reference strain Hardjoprajitno). The reactions of anti-LPS monoclonal antibodies (mAbs) MUM/F1-4/hardjo (IgM) and MUM/F1-6/hardjo (IgG) with whole cell lysates in Western immunoblotting analysis were unaffected by proteinase K treatment. Periodate treatment of the LPS destroyed the binding of MUM/F1-6/hardjo but preserved that of MUM/F1-4/hardjo. Alkaline phosphatase decreased significantly the binding of MUM/F1-4/hardjo to the LPS but only slightly that of MUM/F1-6/hardjo. On the other hand, phosphodiesterase totally destroyed the binding capacity of both monoclonal antibodies in enzyme immunoassays (EIA). A number of mono- and oligosaccharides was used in EIA inhibition studies. Mannose-6-phosphate and galactose-6-phosphate inhibited the binding of MUM/F1-4/hardjo (50% inhibition at a concentration of 5 mM) to the antigen, but glucose-6-phosphate did not. Galactosamine and mannosamine inhibited the binding of MUM/F1-6/hardjo (50% inhibition at a concentration of 3-4 mM), whereas only a weak inhibition was observed with glucosamine. In contrast, N-acetylated amino sugars did not show any inhibition. An O-acetyl group also appears to be involved in the antigen-antibody binding process.

We evaluated levels of mannose-6-phosphate/insulin growth factor-II receptor (M6P/IGFII-R) RNA in 37 breast cancer tumors by quantitative in situ hybridization using a computer-aided image analyzer and compared them to cathepsin D RNA and protein levels in the same tissues. Breast cancer cells expressed more cathepsin D and M6P/IGFII-R RNA than fibroblasts in the same tumors. We found a significant increase of cathepsin D RNA (P = 1 x 10(-5)) and M6P/IGFII-R RNA (P = 0.02) in breast cancer cells compared to epithelial cells of benign mastopathies. There was a positive correlation (r = 0.65; P = 1 x 10(-5)) between M6P/IGFII-R and cathepsin D RNA levels measured on serial sections. This contrasted with the inverse relationship of these 2 RNA species in breast cancer cell lines where estrogen down-regulates M6P/IGFII receptor RNA levels. Moreover, in vivo we found no correlation between the M6P/IGFII-R RNA level and menopausal or estrogen receptor status, suggesting that the in vivo regulation of M6P/IGFII-R RNA differs from its in vitro regulation in cell lines. The M6P/IGFII-R RNA level was not correlated with cathepsin D status, histological grade, and tumor size but was significantly higher in lymph node-positive tumors (P = 0.047). The M6P/IGFII-R could therefore be an additional parameter to predict aggressive breast cancers, complementing cathepsin D assays and other more classical prognostic parameters.

We have developed a broadly applicable solid phase assay for quantitative and qualitative analysis of phosphorylated lysosomal enzymes and other glycoproteins that contain mannose 6-phosphate (Man6-P) residues. Samples are immobilized on membranes and proteins that contain Man6-P are detected using an iodinated, soluble form of the cation-independent Man6-P receptor. The assay is highly specific, sensitive, and linear over 3 orders of magnitude. The technique has two general applications. First, it can be used in a dot blot format to analyze large numbers of samples for total levels of Man6-P glycoproteins. Second, it can be used in a western blot format to identify Man6-P glycoproteins that have been fractionated by polyacrylamide gel electrophoresis. Specific applications of this novel assay include structural characterization of glycoproteins, diagnosis of mucolipidosis II and III, and screening of pharmaceuticals for lysosomotropic activity. Other potential applications are discussed.

Human lysosomal alpha-galactosidase A (alpha-Gal A) was stably overexpressed in CHO cells and its biosynthesis and targeting were investigated. Clone AGA5.3-1000Mx, which was the highest enzyme overexpressor, produced intracellular alpha-Gal A levels of 20,900 U/mg (approximately 100 micrograms of enzyme/10(7) cells) and secreted approximately 13,000 U (or 75 micrograms/10(7) cells) per day. Ultrastructural examination of these cells revealed numerous 0.25-1.5 microns crystalline structures in dilated trans-Golgi network (TGN) and in lysosomes which stained with immunogold particles using affinity-purified anti-human alpha-Gal A antibodies. Pulse-chase studies revealed that approximately 65% of the total enzyme synthesized was secreted, while endogenous CHO lysosomal enzymes were not, indicating that the alpha-Gal A secretion was specific. The recombinant intracellular and secreted enzyme forms were normally processed and phosphorylated; the secreted enzyme had mannose-6-phosphate moieties and bound the immobilized 215-kD mannose-6-phosphate receptor (M6PR). Thus, the overexpressed enzyme's selective secretion did not result from oversaturation of the M6PR-mediated pathway or abnormal binding to the M6PR. Of note, the secreted alpha-Gal A was sulfated and the percent of enzyme sulfation decreased with increasing amplification, presumably due to the inaccessibility of the enzyme's tyrosine residues for the sulfotransferase in the TGN. Overexpression of human lysosomal alpha-N-acetylgalactosaminidase and acid sphingomyelinase in CHO cell lines also resulted in their respective selective secretion. In vitro studies revealed that purified secreted alpha-Gal A was precipitated as a function of enzyme concentration and pH, with 30% of the soluble enzyme being precipitated when 10 mg/ml of enzyme was incubated at pH 5.0. Thus, it is hypothesized that these overexpressed lysosomal enzymes are normally modified until they reach the TGN where the more acidic environment of this compartment causes the formation of soluble and particulate enzyme aggregates. A significant proportion of these enzyme aggregates are unable to bind the M6PR and are selectively secreted via the constitutive secretory pathway, while endogenous lysosomal enzymes bind the M6PRs and are transported to lysosomes.

The transforming growth factor-beta 1 (TGF beta 1) and -beta 2 (414) precursors both contain three predicted sites of N-linked glycosylation within their pro regions. These are located at amino acid residues 72, 140, and 241 for the TGF beta 2 (414) precursor and at residues 82, 136, and 176 for the TGF beta 1 precursor; both proteins contain mannose-6-phosphate (M-6-P) residues. The major sites of M-6-P addition are at Asn (82) and Asn (136), the first two sites of glycosylation, for the TGF beta 1 precursor. We now show that the major site of M-6-P addition within the TGF beta 2 (414) precursor is at Asn241, the third glycosylation site. To determine the importance of N-linked glycosylation to the secretion of TGF beta 1 and -beta 2, site-directed mutagenesis was used to change the Asn residues to Ser residues; the resulting DNAs were transfected into COS cells, and their supernatants were assayed for TGF beta activity. Substitution of Asn (241) of the TGF beta 2 (414) precursor resulted in an 82% decrease in secreted TGF beta 2 bioactivity. Mutation at Asn72 resulted in a 44% decrease, while mutation at Asn140 was without effect. Elimination of all three glycosylation sites resulted in undetectable levels of TGF beta 2. These results were compared with similar mutations made in the cDNA encoding the TGF beta 1 precursor. Mutagenesis of the two M-6-P-containing sites (Asn82 and Asn136) resulted in an 83% decrease in secreted TGF beta 1; replacement of Asn82 and Asn136 with Ser individually resulted in 85% and 42% decreases in activity, respectively. Substitution of Asn176 with Ser was without effect, while substitution of all three sites of glycosylation resulted in undetectable levels of TGF beta 1 activity, similar to the results obtained with TGF beta 2. The nine Cys residues within the mature region of TGF beta 1 were mutated to serine, and their effects on TGF beta 1 secretion were evaluated. Mutation of most Cys residues resulted in undetectable levels of TGF beta 1 protein or activity in conditioned medium. Mutation of Cys (355) led to the secretion of inactive TGF beta 1 monomers, suggesting that this residue is either directly involved in dimer formation or required for correct interchain disulfide bond formation.

Previous studies demonstrated that mannan is a potent inhibitor of splenic entry of lymphocytes and mediates its inhibitory effect at an unidentified site in the spleen rather than acting directly on lymphocytes. This report describes the in vivo site of action of mannan. In vivo localization studies with fluoresceinated preparations of mannan (Fl-mannan) and a mannose-6-phosphate-containing yeast phosphomannan monoester core from P. holstii exopolysaccharide (Fl-PPME) demonstrated that the polysaccharide specifically localize in the splenic marginal sinuses in cells with a dendritic morphology termed splenic sinusoidal cells (SSC). Uptake of the polysaccharides by SSC was mediated by a mannan-specific receptor which was saturable and of high avidity. Several lines of evidence suggested that mannan uptake by SSC inhibited splenic entry of lymphocytes. First, the ability of SSC to bind Fl-mannan and Fl-PPME closely paralleled the ability of these polysaccharides to inhibit splenic entry of lymphocytes. In fact, doses of mannan and PPME which would saturate SSC mannan receptors completely blocked splenic entry of lymphocytes. Second, SSC are situated at the initial entry point of lymphocytes into spleen and passage of lymphocytes through the SSC region of spleen was profoundly inhibited by mannan. Finally, direct evidence for adhesion between lymphocytes and SSC was obtained with spleen cell suspensions where clustering between Fl-mannan labeled SSC and lymphocytes was observed. Collectively, these data indicate that mannan (and PPME) inhibit splenic entry of lymphocytes by interacting with SSC, cell which play a critical role in the entry of lymphocytes into the spleen. Whether mannan-specific receptors on SSC directly mediate lymphocyte-SSC adhesion or play on indirect role in modifying lymphocyte migration requires further investigation.

There are at least three stages in the targeting of soluble lysosomal enzymes: transfer of N-acetylglucosaminyl 1-phosphate to high-mannose oligosaccharide side chains, removal of N-acetylglucosamine and recognition of the "uncovered" mannose 6-phosphate residues. Defects in the transfer reaction cause mucolipidoses II and III. Those in the subsequent stages of the targeting may result in similar clinical disorders. To differentiate between possible defects of the targeting in cultured cells we have developed a procedure for a combined detection of the phosphorylation, uncovering of the transferred phosphate residues and the targeting of lysosomal enzymes. For this purpose cultured cells are metabolically labelled with [32P]phosphate and a lysosomal enzyme, such as cathepsin D, is isolated from the labelled cells and the medium by immunoprecipitation. The immunoprecipitates are dissolved with sodium dodecylsulphate and incubated in the presence and absence of calf intestine alkaline phosphatase. We show that the treatment of the denatured protein results in hydrolysis of phosphomonoester groups and that the phosphodiester and the peptide bonds remain intact. The initial and the residual radioactivity associated with the lysosomal enzyme which represent the total phosphate and the phosphodiester groups, respectively, are determined by gel-electrophoresis, fluorography and densitometry. This procedure extends one of the previously established methods for the diagnosis of mucolipidoses II and III.

The size of a lysosomal cysteine proteinase from epimastigotes of Trypanosoma cruzi decreased from 60 to 54 kDa upon treatment with endo beta-N-acetylglucosaminidase H. A lower-molecular weight component (30-35 kDa), which usually accompanies the 60-kDa protein also increased its electrophoretic mobility, and seems to consist of a mixture of degradation products of the enzyme, since both the larger and the smaller components had the same N-terminal sequence as the 60-kDa protein. The amino acid composition of the protein moiety and the composition of the oligosaccharide chains have been determined. The oligosaccharide chains are of the high-mannose type, and contain 6, 7, 8 or 9 mannose residues, as shown both by in vivo labeling with [U-14C]glucose, and by labeling the endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides of the purified enzyme with tritiated sodium borohydride. The oligosaccharide chains did not contain phosphate residues. Further studies with [U-14C]-labeled total glycoproteins of T. cruzi and enzyme assays, suggest that T. cruzi and other trypanosomatids do not target their lysosomal enzymes to the organelle through the mannose-6-phosphate marker pathway.

Chinese hamster ovary (CHO) clones secreting high levels of transforming growth factor-beta 2 (TGF-beta 2) were obtained after transfection with a cDNA clone coding for the 414-amino acid TGF-beta 2 precursor and subsequent amplification with methotrexate. The TGF-beta 2 was secreted in a latent form since acidification was necessary for detection of maximal levels of bioactivity. Amino- and carboxy-terminal sequencing of purified recombinant TGF-beta 2 indicated that correct processing of mature TGF-beta 2 had occurred. In addition to mature TGF-beta 2, the recombinant CHO clones secreted larger proteins having molecular weights of 85, 105, and 130 kD, which consisted of both mature and pro-region sequences when analyzed by immunoblotting using site-specific anti-peptide antibodies. Analysis of serum- and cell-free media from recombinant CHO cells metabolically labeled with [3H]glucosamine and [32P]orthophosphate indicated that pro-TGF-beta 2 was glycosylated and phosphorylated. Two-dimensional electrophoretic analysis of acid hydrolysates showed that the 32P was incorporated into mannose-6-phosphate.

Uteroferrin is an iron-containing, progesterone-induced, acid phosphatase that is secreted in large amounts by the uterine endometrium of pigs. During pregnancy, it transports iron across the chorioallantois (placenta) for use in fetal hematopoiesis. In this paper, it is reported that uteroferrin synthesized by cultured endometrial explants possesses N-linked, high-mannose, oligosaccharide chains that contain 6- phosphomannose units. The latter is regarded as a possible recognition marker whereby acid hydrolases are targeted to the lysosome. On uteroferrin, however, the majority of the phosphate is in single diester linkages between the mannose and a covering N-acetylglucosamine. It is suggested that uteroferrin is a lysosomal enzyme that has assumed a role in iron transport and metabolism and is secreted because the covering N-acetylglucosamine is not removed.

Recombinant transforming growth factor-beta 1 (TGF-beta 1) precursor produced and secreted by a clone of Chinese hamster ovary cells was found to be glycosylated and phosphorylated. Treatment of 32P-labeled precursor protein with N-glycanase indicated that phosphate was incorporated into asparagine-linked complex carbohydrate moieties. Fractionation of 32P-labeled glycopeptides followed by amino acid sequence analysis indicated that greater than 95% of the label was incorporated into two out of three glycosylation sites at Asn-82 and Asn-136 of the TGF-beta 1 precursor. Two-dimensional electrophoretic analysis of acid hydrolyzed precursor protein and precursor protein-derived glycopeptides indicated that 32P was incorporated as mannose 6-phosphate. Binding studies with the purified receptor for mannose 6-phosphate indicated that the TGF-beta 1 precursor could bind to this receptor and the binding was specifically inhibited with mannose 6-phosphate.

Interaction of mannose 6-phosphate-specific receptors with their ligands has been suggested to be essential for natural killer cell (NK)-mediated cytotoxicity. Indeed, mannose 6-phosphate-specific receptors and ligands bearing mannose 6-phosphate residues are demonstrable on human peripheral blood leukocytes with NK activity as well as on K-562 NK target cells, allowing at least in principle such an interaction. It can also be shown that NK activity of human peripheral blood leukocytes is inhibited by mannose 6-phosphate. The following observations, however, exclude an essential role of the mannose 6-phosphate receptor-ligand system in NK cell-mediated cytotoxicity. 1) NK cytotoxicity is sensitive to a broad range of structurally unrelated sugar phosphates. 2) NK activity is normal in patients with I cell disease (mucolipidosis II), which due to a genetic defect are unable to synthesize the ligands for the mannose 6-phosphate-specific receptor. 3) NK cytotoxicity is not inhibited by an antiserum against the mannose 6-phosphate receptor, which blocks the receptor function.

Yeast cell wall mannoproteins often contain phosphate esterified to the oligosaccharide side chains, and partial acetolysis yields both neutral and phosphorylated fragments [Thieme, T. R., & Ballou, C. E. (1971) Biochemistry 10, 4121--4129]. We have isolated the acidic acetolysis fragments from Hansenula wingei mannoprotein [Yen, P. H., & Ballou, C. E. (1974) Biochemistry 13, 2420--2427] and have separated them into a mannopentaose monophosphate (Man5P) and a mannotriose monophosphate (Man3P). On the basis of selective enzymatic and partial acid hydrolysis and 1H and 31P NMR studies, Man5P was shown to have the structure P leads to 6 alpha Man1 leads to 3 alpha Man1 leads to 2 alpha Man1 leads to 2 alpha Man1 leads to 2Man (where Man = D-mannopyranose). The 31P NMR spectrum of the Man3P showed it to be a mixture of a phosphate monoester and a phosphate diester, which was separated by ion-exchange chromatography. The monoester had the structure P leads to 6 alpha Man1 leads to 3 alpha Man1 leads to 3Man whereas the diester had the properties of a cyclic phosphate. Although native H. wingei mannoprotein contains phosphodiester linkages, the starting mannoprotein preparation was isolated under alkaline conditions that hydrolyze such bonds, and it did not show a phosphodiester signal in the 31P NMR spectrum. We conclude that the cyclic phosphate was an artifact formed during the acetolysis reaction. Because acetolysis of H. wingei mannoprotein yields only phosphorylated mannotriose and mannopentaose whereas the mannoprotein contains mannotetraose side chains as well, the phosphorylation process must be a very specific event in the biosynthesis of the glycoprotein.

Lysosomal enzymes isolated from the slime mold Dictyostelium discoideum bind to the mannose 6-phosphate receptor which is present in many mammalian cells. While binding to the receptor suggests that the slime mold enzymes possess the same mannose 6-phosphate recognition marker as their mammalian counterparts, initial structural studies of the phosphorylated oligosaccharides have indicated that the phosphate is attached to high mannose-type units via an unusual phosphodiester linkage (Freeze, H.H., Yeh, R., Miller, A.L., and Kornfeld, S. (1983) J. Biol. Chem. 258, 14874-14879). To identify the components of the phosphodiester group we have isolated the phosphorylated high-mannose oligosaccharides from D. discoideum AX3 cells labeled with [2-3H]mannose or [6-3H]glucosamine and from the differentiation medium of unlabeled cells. The major phosphorylated species contain one or two phosphodiester groups and an average of 6 or 7 mannose residues. The phosphodiesters are relatively stable to both acid and base hydrolysis, but upon strong acid hydrolysis (conditions that completely hydrolyze the oligosaccharide) mannose 6-phosphate residues are liberated. Through a combination of techniques, including fast atom bombardment and direct chemical ionization mass spectrometry, it is shown that the mannose 6-phosphate residues of the intact oligosaccharide are diesterified to methyl groups. This indicates that slime mold possesses a different biosynthetic pathway for the formation of phosphorylated high mannose-type oligosaccharides than is utilized by higher organisms.

Phosphomannosyl residues on acid hydrolases serve as recognition markers which target these enzymes to lysosomes. We have found that the oligosaccharide units of newly synthesized beta-glucuronidase contain phosphate residues in diester linkage between mannose and alpha-linked N-acetylglucosamine residues (Tabas, I., and Kornfeld, S. (1980) J. Biol. Chem. 255, 6633--6639). To obtain larger amounts of these molecules for structural studies, total cellular glycopeptides were isolated from [2-3H]mannose-labeled mouse lymphoma cells, and the phosphorylated oligosaccharides were released by endo-beta-N-acetylglucosaminidase CII and H and isolated by gel filtration and ion exchange chromatography. The fractions were characterized by alpha-mannosidase digestion before and after removal of the phosphate residues and by acetolysis. We also determined whether the phosphate was present as a phosphomonoester or as a diester. The phosphorylated oligosaccharides consisted of a family or related molecules, all of which contained a high mannose-type oligosaccharide core. The major class consisted of isomers containing a single phosphate in diester linkage to 1 of 3 mannose residues of the underlying oligosaccharide. The second class contained isomers with two phosphodiester groups located at five different positions of the oligosaccharide. In both of these classes, the phosphodiester group could be converted to a phosphomonoester by pig liver alpha-N-acetylglucosaminidase, indicating that the cover is alpha-linked N-acetylglucosamine. The third class was similar to the second except that the 2 phosphate residues were present as monoester groups. The last class contained molecules with a single phosphomonoester group. These molecules differed from those with single phosphodiester groups in that the core oligosaccharides were smaller. This is consistent with their being more mature species which have undergone partial processing. These data demonstrate that phosphorylation can occur at 5 separate mannose residues on the high mannose-type oligosaccharides. Individual molecules can have 1, 2, and perhaps even 3 phosphate residues. The majority of the newly synthesized phosphorylated oligosaccharides contain phosphate groups in diester linkage.

A ceramide octasaccharide containing mannose-6-phosphate was isolated from the fresh-water bivalve Corbicula sandai by solvent fractionation, followed by two types of silicic acid column chromatography, and finally QAE-Sephadex column chromatography. The structural analysis involved the following steps. (a) Gas-liquid chromatography of the component sugars, fatty acids, and long-chain bases. (b) Degradation with HCl and HF to elucidate the sugar sequence. (c) Permethylation analysis coupled with GC-MS to identify the positions of the glycosidic linkages between the sugar units. (d) Chromium trioxide oxidation to determine the anomeric configuration. (e) Smith degradation to determine the site of linkage of the ethanolamine residue. The structure of this novel glycolipid was determined to be: 4-O-MeGalp(bets1 yields 3)-GalNAcp(beta1 yields3)Fucp(alpha1 yields 4)GlcNAcp(beta1 yields 2)Manp(alpha1 yields 3)[Xylp(alpha1 yields 2)][2'-aminoethylphosphoryl(yields 6)]Manp(beta1yields 4)Glcp(beta1 yields 1)ceramide. It is very interesting that fucose was found to be internally linked in this sugar chain. To our knowledge, this is the first example of internal fucose in a glycolipid. The ceramide moiety consisted of normal saturated fatty acids, among which stearic acid was pr

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.

Carboxypeptidase Y, a vacuolar enzyme from Saccharomyces cerevisiae, was digested with endo-beta-N-acetyl-D-glucosaminidase H to release the four oligosaccharide chains that are linked to asparagine in the glycoprotein. The oligosaccharides were fractionated into a neutral and acidic component, and the latter proved to phosphorylated. From its gel filtration pattern, the neutral fraction was shown to be a mixture of at least four homologs, the smallest of which had a proton NMR spectrum almost identical to that given by an IgM oligosaccharide with eight mannoses and one N-acetylglucosamine [Cohen, R. E. & Ballou, C. E. (1980) Biochemistry 19, 4345--4358]. The yeast oligosaccharide has one additional mannose unit in an alpha 1 leads to 3 or alpha 1 leads to 6 linkage, whereas the larger homologs appear to have two, three, and four more mannose units. One phosphorylated oligosaccharides with a mannose/phosphate ratio of 12.5 was reduced with NaB3H4 and then subjected to mild acid hydrolysis. This released mannose and mannobiose that were glycosidically linked to the phosphate group, whereas complete acid hydrolysis yielded D-mannose 6-phosphate. The recovered oligosaccharide phosphomonoester, which contained 11 or 12 mannose units, was digested exhaustively with alpha-mannosidase, and the product of this reaction was treated with alkaline phosphatase, which yielded radioactive Man3GlcNAcH2. These results suggest that the mannosidase-resistant phosphorylated oligosaccharide has the structure Man leads to P leads to 6 alpha Man leads to alpha Man leads to 6 beta Man leads to 4GlcNAcH2, in which some of the phosphate groups are substituted with mannobiose instead of mannose. A second phosphorylated oligosaccharide with a mannose/phosphate ratio of 6.5 probably contains two phosphodiester groups, but its structure has not been investigated in detail.

Bovine testicular beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) is rapidly and selectively assimilated by human skin fibroblasts. The assimilation of the enzyme is strongly inhibited by mannose 6-phosphate and by a glycoprotein fraction isolated from bovine testes (glycoprotein inhibitors). These results suggest that beta-galactosidase and the glycoprotein inhibitors have a common recognition marker that contains mannose 6-phosphate. The presence of mannose phosphate in the glycoprotein inhibitors was demonstrated by acid hydrolysis of the glycoproteins to liberate mannose phosphate followed by reduction with NaB(3)H(4) to give [(3)H]mannitol phosphate. The (3)H-labeled compound was identified by paper electrophoresis and by the release of [(3)H]mannitol on treatment with phosphatase. The [(3)H]mannitol phosphate was oxidized with periodate and the resulting phosphorylated fragment, on reduction with NaB(3)H(4), yielded [(3)H]ethylene glycol phosphate, indicating substitution of phosphate on carbon 6 of mannitol. Mannose 6-phosphate was also found in a major carbohydrate-containing fraction of peptides produced from the glycoprotein inhibitors by tryspin digestion. It was estimated that about 2% of the mannose residues were present as mannose 6-phosphate. Phosphorylated oligosaccharides were also identified in hydrolysates of the glycoprotein inhibitors. One, a disaccharide, was identified as alpha-(mannosyl-6-phosphate)-(1 --> 2)-mannose. These observations suggest that the recognition marker of beta-galactosidase contains alpha1,2-linked mannose 6-phosphate; terminal alpha1,2-linked mannose residues are known to occur in the high-mannose type oligosaccharides present on beta-galactosidase.

Phosphorylated and nonphosphorylated tri- and tetrasaccharides were isolated from yeast mannan and analyzed by 500-MHz 1H NMR spectroscopy. Relayed spin-echo difference spectroscopy (RESED) was used to reveal subspectra of the phosphorylated residues. In this way the attachment position of the phosphate group could be established. The structures of the phosphorylated compounds turned out to be (Formula: see text) and (Formula: see text) RESED spectroscopy has proved to be a suitable method in analyzing low amounts of phosphorylated oligosaccharides.

A significant elevation of cathepsin D activity was observed in six human hepatoma tissues as compared to 12 normal human livers. In isoelectric focusing experiments, cathepsin D purified from normal liver exhibited three different forms, with isoelectric points of 5.6, 6.1, and 6.7, while cathepsin D purified from hepatoma contained another five to six more acidic forms in addition to the forms observed in normal liver cathepsin D. When the tumor enzyme was treated with endo-beta-N-acetylglucosaminidase H followed by isoelectric focusing, the acidic components disappeared and were converted to forms identical to those of the normal liver cathepsin D. Determination of the mannose-6-phosphate content showed that hepatoma cathepsin D contains twice as much mannose-6-phosphate as normal liver cathepsin D. Peptide mapping and amino acid analysis showed that the protein moiety of cathepsin D from hepatoma is almost identical with that from normal liver. These findings indicate that the appearance of acidic variants in hepatoma cathepsin D is mainly due to changes in the oligosaccharide chains of the enzyme, which are closely associated with the increase of mannose-6-phosphate in the tumor enzyme.

In human fibroblasts, the recognition of lysosomal enzymes by cell surface receptors is mediated by mannose 6-phosphate residues located on oligosaccharides that can be cleaved by endo-beta-N-acetylglucosaminidase H. About half of these oligosaccharides, as isolated from beta-hexosaminidase and cathepsin D secreted by human skin fibroblasts, are anionic. Most of these are resistant to alkaline phosphatase. The resistance is due to alpha-N-acetylglucosamine residues linked to mannose 6-phosphate by a phosphodiester bond. The major phosphorylated oligosaccharides contain one and two and possibly three phosphate groups blocked by N-acetylglucosamine. Besides the blocked phosphate groups these oligosaccharides contain a common inner core consisting of Man alpha 1,6-(Man alpha 1,3)Man alpha 1,6(Man alpha 1,3)Man beta GlcNAc and either one or two alpha 1,2-linked mannose residues.

Acid hydrolases from mammalian sources contain phosphorylated oligosaccharides which function as recognition markers for their receptor-mediated endocytosis by human fibroblasts. The discovery that glycopeptides derived from the slime mold, Dictyostelium discoideum, contain mannose 6-phosphate led to the suggestion that acid hydrolases from this source might also bear the marker. To test this hypothesis, the binding and endocytosis of purified beta-D-glucosidase (EC 3.2.1.21), beta-N-acetyl-D-hexosaminidase (EC 3.2.1.52), and alpha-D-mannosidase (EC 3.2.1.24) by human fibroblasts were investigated. These enzymes underwent endocytosis with efficiencies of 8.6 to 60%/mg/h, and 1 mM mannose 6-phosphate markedly inhibited their uptake (80 to 100%). The specificity of inhibition by sugar phosphates, the saturation kinetics of endocytosis, and the binding properties of D. discoideum acid hydrolases were similar to those reported for enzyme preparations derived from mammalian sources. In addition, 95 to 100% of the beta-D-glucosidase or alpha-D-mannosidase molecules from D. discoideum preparations were competent for in vitro clearance. Furthermore, the three purified acid hydrolases contain 5 to 7 mol of mannose 6-phosphate/mol of enzyme. This indicates that, unlike many mammalian enzyme preparations, most if not all of these enzyme molecules from D. discoideum contain the phosphomannosyl recognition marker.

Thyroglobulin (TG), the major exportable protein of thyroid follicle cells, is conveyed to lysosomes on a complex secretion, storage and recapture pathway by as yet unknown transport mechanisms. This report establishes that the dimeric porcine TG-molecule carries an average of six phosphate residues. Endoglycosidase digestion showed that two phosphate residues are bound to the high-mannose carbohydrate side chains (CHO), while two others are linked to the complex CHO. These four residues are also sensitive to alkaline phosphatase treatment, indicating their terminal linkage. Immunoprecipitation analyses showed that TG obtained from microsomal fractions is already phosphorylated. Most important, an enzymatic assay applied to hydrolysates of TG established that the two phosphate residues at the high mannose CHO are present as mannose-6-phosphate (M-6-P). Alkaline phosphatase treatment of biosynthetically radiophosphorylated CHO followed by hydrolysis and t.l.c. indicated that M-6-P is present at least in part in phosphomonoester linkage. Furthermore, porcine TG binds specifically to the M-6-P receptor of Chinese hamster ovary cells. It is concluded that the M-6-P residues of TG are exposed and able to operate as a ligand for the M-6-P receptor. It is unknown why the lysosomal recognition-marker M-6-P does not convey TG directly on an intracellular route to lysosomes. We propose that for the secretion of newly synthesized TG into the follicle lumen an additional export signal dominating over the M-6-P recognition-marker is required.