In vivo clearance and metabolism of recombinant activated factor VII (rFVIIa) and its complexes with plasma protease inhibitors in the liver.

INTRODUCTION: Recombinant activated factor VII (rFVIIa, NovoSeven®) is injected intravenously for the treatment of haemophilia patients with inhibitory antibodies. In plasma, rFVIIa forms complexes with protease inhibitors, primarily antithrombin III (ATIII). The liver is believed to be involved in clearance of rFVIIa, however, it is not known whether the liver is also involved for the clearance of the rFVIIa-ATIII complex. In this study, we explored the fate of intravenously injected rFVIIa from plasma to the hepatic lysosomes. MATERIALS AND METHODS: A novel method using magnetic chromatography was used to isolate catabolic organelle (CO) fractions from mouse liver following injection of superparamagnetic dextran (SPD)-coated iron oxide particles and rFVIIa. The effect of co-circulating SPD particles on rFVIIa pharmacokinetic (PK) parameters was evaluated by ELISA. Cryo-immuno transmission electron microscopy (TEM) was used to study hepatic distribution of SPD particles and rFVIIa. The isolated hepatic CO fractions were characterized using Western Blotting (WB). RESULTS: Cryo-immuno TEM of the liver confirmed hepatic co-localisation of SPD particles and rFVIIa in identical endosomes and lysosomes of both hepatocytes and Kupffer cells. SPD particles did not affect the PK parameters of rFVIIa. WB analysis of plasma and CO fractions detected rFVIIa as the full-length protein and also in high molecular weight (HMW) complexes with ATIII and α-2 macroglobulin (α-2M). CONCLUSIONS: Following injection, both hepatocytes and Kupffer cells appeared to be involved in the hepatic clearance and metabolism of both full-length rFVIIa and rFVIIa in complex with at least two plasma protease inhibitors; ATIII and α-2M.

The NPC1 protein: structure implies function.

Niemann-Pick type C (NPC) is a lysosomal storage disorder, characterized by intracellular accumulation of low-density lipoprotein (LDL)-derived cholesterol and neurodegeneration leading to premature death. The most common form of the disease, NPC1, results from mutations in the NPC1 gene. Thus, the NPC1 protein is the focus of intense investigation to elucidate the function of this protein and its role in the disease pathogenesis. Recent studies have revealed the NPC1 subcellular location, topology and potential functions of the NPC1 protein. In lieu of direct experimental evidence, certain hypotheses about the function of NPC1 can be inferred by analyzing disease-causing mutations, NPC1 protein sequence homology to other related proteins, and the potential tertiary structure similarity between NPC1 and its prokaryotic ancestors, such as the E. coli RND permease AcrB. This review will discuss recent work on the characterization and function of the NPC1 protein and highlight structural features that may be important in assisting in the elucidation of NPC1 function and role in subcellular lipid transport and homeostasis.

Apoptosis-induced release of mature sterol regulatory element-binding proteins activates sterol-responsive genes.

It is well established that during the execution of the apoptotic cascade, activated caspase 3 releases sterol regulatory element-binding proteins (SREBP) from the membrane of the endoplasmic reticulum in a proteolytic reaction that is distinct from their normal sterol-dependent activation. However, it is not known whether these transcription factors are capable of activating sterol-responsive genes under such conditions. The construction of SRE expression vectors has permitted characterization of the apoptotic activation of SREBP. Cell lines stably expressing the plasma membrane marker CD32, or GFP, under the control of the SRE promoter were shown to modulate SRE gene expression on the basis of the levels of available sterols. However, during the induction of apoptosis, expression of CD32 and GFP was highly induced, even in the presence of ample sterols. Apoptotic induction of sterol-regulated genes was due to activation of caspase 3 and was impervious to treatment with sphingomyelinase, indicating that activation of SRE genes during apoptosis is sterol independent. Further characterization of this apoptotic response indicated that sterol-regulated genes are activated at an early stage in the apoptotic cascade, preceding the externalization of phosphatidylserine on the plasma membrane of apoptotic cells. These results suggest that activation of sterol-responsive genes early during apoptosis may play a role in the proper execution of this program.

Accumulation of cholera toxin and GM1 ganglioside in the early endosome of Niemann-Pick C1-deficient cells.

We investigated intracellular trafficking of GM1 ganglioside in Niemann-Pick C1 (NPC1)-deficient Chinese hamster ovary cells [NPC1(-) cells] by using cholera toxin (CT) as a probe. Both the holotoxin and the B subunit (CTB) accumulated in GM1-enriched intracellular vesicles of NPC1(-) cells. CTB-labeled vesicles contained the early endosome marker Rab5 but not lysosome-associated membrane protein 2 and were not labeled with either Texas red-transferrin or Lysotracker, indicating that they represent early endosomes. Similarly, CT accumulated in intracellular vesicles of human NPC fibroblasts that contained both Rab5 and early endosomal antigen 1. CTB accumulation in NPC1(-) cells was abolished by expression of wild-type NPC1 but not by mutant proteins with a mutation either in the NPC domain or the sterol-sensing domain. A part of these mutant NPC1 proteins expressed in NPC1(-) cells was localized on CTB-labeled vesicles. U18666A treatment of "knock in" cells [NPC1(-) cells that stably expressed wild-type NPC1] caused CTB accumulation similar to that in NPC1(-) cells, and a part of wild-type NPC1was localized on CTB-labeled vesicles in drug-treated cells. Finally, CT tracer experiments in NPC1(-) cells revealed retarded excretion of internalized toxin into the culture medium and an increase in the intracellular release of A subunits. In accordance with the latter result, CT was more effective in stimulating cAMP formation in NPC1(-) than in wild-type cells. These results suggest that transport of CT/GM1 complexes from the early endosome to the plasma membrane depends on the function of NPC1, whereas transport to the Golgi apparatus/endoplasmic reticulum does not.

Multidrug permeases and subcellular cholesterol transport.

Studies of Niemann-Pick C (NPC) and Tangier diseases have led to the identification of the causative genes, NPC1 and ABCA1, respectively. Characterization of their protein products shows that NPC1 and ABCA1 are permeases that belong to two different superfamilies of efflux pumps, which might be important in subcellular lipid and cholesterol transport.

Fabry disease: preclinical studies demonstrate the effectiveness of alpha-galactosidase A replacement in enzyme-deficient mice.

Preclinical studies of enzyme-replacement therapy for Fabry disease (deficient alpha-galactosidase A [alpha-Gal A] activity) were performed in alpha-Gal A-deficient mice. The pharmacokinetics and biodistributions were determined for four recombinant human alpha-Gal A glycoforms, which differed in sialic acid and mannose-6-phosphate content. The plasma half-lives of the glycoforms were approximately 2-5 min, with the more sialylated glycoforms circulating longer. After intravenous doses of 1 or 10 mg/kg body weight were administered, each glycoform was primarily recovered in the liver, with detectable activity in other tissues but not in the brain. Normal or greater activity levels were reconstituted in various tissues after repeated doses (10 mg/kg every other day for eight doses) of the highly sialylated AGA-1 glycoform; 4 d later, enzyme activity was retained in the liver and spleen at levels that were, respectively, 30% and 10% of that recovered 1 h postinjection. Importantly, the globotriaosylceramide (GL-3) substrate was depleted in various tissues and plasma in a dose-dependent manner. A single or repeated doses (every 48 h for eight doses) of AGA-1 at 0.3-10.0 mg/kg cleared hepatic GL-3, whereas higher doses were required for depletion of GL-3 in other tissues. After a single dose of 3 mg/kg, hepatic GL-3 was cleared for > or =4 wk, whereas cardiac and splenic GL-3 reaccumulated at 3 wk to approximately 30% and approximately 10% of pretreatment levels, respectively. Ultrastructural studies demonstrated reduced GL-3 storage posttreatment. These preclinical animal studies demonstrate the dose-dependent clearance of tissue and plasma GL-3 by administered alpha-Gal A, thereby providing the in vivo rationale-and the critical pharmacokinetic and pharmacodynamic data-for the design of enzyme-replacement trials in patients with Fabry disease.

Transmembrane molecular pump activity of Niemann-Pick C1 protein.

Niemann-Pick C1 (NPC1) disease is characterized by cholesterol accumulation in lysosomes and aberrant feedback regulation of cellular cholesterol homeostasis. We provide evidence that the NPC1 protein has homology with the resistance-nodulation-division (RND) family of prokaryotic permeases and may normally function as a transmembrane efflux pump. Studies of acriflavine loading in normal and NPC1 fibroblasts indicated that NPC1 uses a proton motive force to remove accumulated acriflavine from the endosomal/lysosomal system. Expression of NPC1 in Escherichia coli (i) facilitated the transport of acriflavine across the plasma membrane, causing cytosolic accumulation, and (ii) resulted in transport of oleic acid but not cholesterol or cholesterol-oleate across the plasma membrane. These studies establish NPC1 as a eukaryotic member of the RND permease family.

The structure and function of the Niemann-Pick C1 protein.

Niemann-Pick C (NPC) disease is a recessive cholesterol storage disorder characterized by severe, progressive neurodegeneration. The primary causative gene found on chromosome 18q11-12 was identified by a positional cloning approach. The NPC1 gene product is predicted to be a large polytopic glycoprotein with a cytoplasmic tail containing a dileucine endosome-targeting motif. The NPC1 protein sequence shares strong homology with a newly identified homologue, NPC1L1, and the morphogen receptor Patched. In addition, a group of five NPC1 transmembrane domains share homology with the sterol-sensing domain of proteins involved in cellular cholesterol homeostasis. Subcellular localization studies have shown NPC1 to reside in late endosomes and to transiently associate with lysosomes and the trans-Golgi network. Analysis of its topological arrangement in membranes suggests that NPC1 contains 13 transmembrane domains and three large, hydrophilic, lumenal loops. Currently, there is no direct evidence as to the function of the NPC1 protein; however, a number of observations suggest that NPC1 may be related to a family of prokaryotic efflux pumps and thus it may also act as a molecular pump.

Topological analysis of Niemann-Pick C1 protein reveals that the membrane orientation of the putative sterol-sensing domain is identical to those of 3-hydroxy-3-methylglutaryl-CoA reductase and sterol regulatory element binding protein cleavage-activating protein.

The Niemann-Pick C1 (NPC1) protein is predicted to be a polytopic glycoprotein, and it contains a region with extensive homology to the sterol-sensing domains (SSD) of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-R) and sterol regulatory element binding protein cleavage-activating protein (SCAP). To aid the functional characterization of NPC1, a model of NPC1 topology was evaluated by expression of epitope-tagged NPC1 proteins and investigation of epitope accessibility in selectively permeabilized cells. These results were further confirmed by expression of NPC1 and identification of glycosylated domains that are located in the lumen of the endoplasmic reticulum. Our data indicate that this glycoprotein contains 13 transmembrane domains, 3 large and 4 small luminal loops, 6 small cytoplasmic loops, and a cytoplasmic tail. Furthermore, our data show that the putative SSD of NPC1 is oriented in the same manner as those of HMG-R and SCAP, providing strong evidence that this domain is functionally important.

Evidence for a Niemann-pick C (NPC) gene family: identification and characterization of NPC1L1.

Niemann-Pick type C1 (NPC1) disease is caused by defects in the NPC1 protein, which result in perturbation of subcellular cholesterol transport. To identify related proteins that may be involved in subcellular cholesterol trafficking, the expressed sequence tag (EST) database was searched to find homologues of human NPC1. A short, weakly similar EST was identified and used to obtain a full-length human cDNA of about 5 kb and two alternatively spliced transcripts. The gene, named NPC1L1, was mapped to chromosome 7p13, contained 20 exons, including an unusually large 1526-bp exon 2, and spanned approximately 29 kb. In contrast to NPC1, the NPC1L1 putative promoter region contained a sterol-regulatory element. The predicted protein shared 42% identity and 51% similarity with NPC1. Interestingly, NPC1L1 contains the conserved amino-terminal "NPC1 domain" and the putative sterol-sensing domain, providing strong evidence that it is related to human NPC1 and suggesting that these may comprise a new family of NPC1-related proteins. However, the two differ with respect to their putative intracellular targeting signals. Collectively, these data suggest that NPC1L1 and NPC1 form part of a family of related proteins that may have similar functions at different subcellular locations, perhaps at sequential steps of the same cholesterol transport pathway.

Human alpha-N-acetylgalactosaminidase: site occupancy and structure of N-linked oligosaccharides.

Human alpha-N-acetylgalactosaminidase (alpha-GalNAc; also known as alpha-galactosidase B) is the lysosomal exoglycohydrolase that cleaves alpha-N-acetylgalactosaminyl moieties in glycoconjugates. Mutagenesis studies indicated that the first five (N124, N177, N201, N359, and N385) of the six potential N-glycosylation sites were occupied. Site 3 occupancy was important for enzyme function and stability. Characterization of the N-linked oligosaccharide structures on the secreted enzyme overexpressed in Chinese hamster ovary cells revealed highly heterogeneous structures consisting of complex (approximately 53%), hybrid (approximately 12%), and high mannose-type (approximately 33%) oligosaccharides. The complex structures were mono-, bi-, 2,4-tri-, 2,6-tri-, and tetraantennary, among which the biantennary structures were most predominant (approximately 53%). Approximately 80% of the complex oligo-saccharides had a core-region fucose and 50% of the complex oligosaccharides were sialylated exclusively with alpha-2,3-linked sialic acid residues. The majority of hybrid type oligo-saccharides were GalGlcNAcMan(6)GlcNAc-Fuc(0-1)GlcNAc. Approximately 54% of the hybrid oligosaccharide were phosphorylated and one-third of these structures were further sialylated, the latter representing unique phosphorylated and sialylated structures. Of the high mannose oligosaccharides, Man(5-7)GlcNAc(2) were the predominant species (approximately 90%) and about 50% of the high mannose oligosaccharides were phosphorylated, exclusively as monoesters whose positions were determined. Comparison of the oligosaccharide structures of alpha-GalNAc and alpha-galactosidase A, an evolutionary-related and highly homologous exoglycosidase, indicated that alpha-GalNAc had more completed complex chains, presumably due to differences in enzyme structure/domains, rate of biosynthesis, and/or aggregation of the overexpressed recombinant enzymes.

Niemann-Pick C1 is a late endosome-resident protein that transiently associates with lysosomes and the trans-Golgi network.

Niemann-Pick type C (NPC) disease is a severe cell lipidosis characterized by the accumulation of unesterified cholesterol in the endosomal/lysosomal system. Recently the primary disease-causing gene, NPC1, was identified, but few clues regarding its potential function(s) could be derived from its predicted amino acid sequence. Therefore, efforts were directed at characterizing the subcellular location of the NPC1 protein. Initial studies with a FLAG-tagged NPC1 cDNA demonstrated that NPC1 is a glycoprotein that associates with the membranes of a population of cytoplasmic vesicles. Immunofluorescence microscopy using anti-NPC1 polyclonal antibodies confirmed this analysis. Double-label immunofluorescence microscopy and subcellular fractionation studies indicated that NPC1 associates predominantly with late endosomes (Rab9 GTPase-positive vesicles) and, to a lesser extent, with lysosomes and the trans-Golgi network. When cholesterol egress from lysosomes was blocked by treatment of cells with U18666A, the NPC1 location shifted from late endosomes to the trans-Golgi network and lysosomes. Subcellular fractionation of liver homogenates from U18666A-treated mice confirmed these observations. These data suggest that U18666A may inhibit the retrograde transport of NPC1 from lysosomes to late endosomes for subsequent transfer to the trans-Golgi network.

Correction of enzymatic and lysosomal storage defects in Fabry mice by adenovirus-mediated gene transfer.

Fabry disease is a recessive, X-linked disorder caused by a deficiency of the lysosomal hydrolase alpha-galactosidase A. Deficiency of this enzyme results in progressive deposition of the glycosphingolipid globotriaosylceramide (GL-3) in the vascular lysosomes, with resultant distension of the organelle. The demonstration of a secretory pathway for lysosomal enzymes and their subsequent recapture by distant cells through the mannose 6-phosphate receptor pathway has provided a rationale for somatic gene therapy of lysosomal storage disorders. Toward this end, recombinant adenoviral vectors encoding human alpha-galactosidase A (Ad2/CEHalpha-Gal, Ad2/CMVHIalpha-Gal) were constructed and injected intravenously into Fabry knockout mice. Administration of Ad2/CEHalpha-Gal to the Fabry mice resulted in an elevation of alpha-galactosidase A activity in all tissues, including the liver, lung, kidney, heart, spleen, and muscle, to levels above those observed in normal animals. However, enzymatic expression declined rapidly such that by 12 weeks, only 10% of the activity observed on day 3 remained. Alpha-galactosidase A detected in the plasma of injected animals was in a form that was internalized by Fabry fibroblasts grown in culture. Such internalization occurred via the mannose 6-phosphate receptors. Importantly, concomitant with the increase in enzyme activity was a significant reduction in GL-3 content in all tissues to near normal levels for up to 6 months posttreatment. However, as expression of alpha-galactosidase A declined, low levels of GL-3 reaccumulated in some of the tissues at 6 months. For protracted treatment, we showed that readministration of recombinant adenovirus vectors could be facilitated by transient immunosuppression using a monoclonal antibody against CD40 ligand (MR1). Together, these data demonstrate that the defects in alpha-galactosidase A activity and lysosomal storage of GL-3 in Fabry mice can be corrected by adenovirus-mediated gene transfer. This suggests that gene replacement therapy represents a viable approach for the treatment of Fabry disease and potentially other lysosomal storage disorders.

Quantitative determination of globotriaosylceramide by immunodetection of glycolipid-bound recombinant verotoxin B subunit.

An assay for the neutral glycosphingolipid, globotriaosylceramide (Galalpha1-4Galbeta1-4Glcbeta1-1Cer; GL-3), was developed based on the B subunit of Escherichia coli verotoxin (VTB). The VTB gene was isolated, overexpressed in E. coli, and purified by a single immunoaffinity chromatographic step using a monoclonal anti-VTB IgG-agarose column. Purified recombinant VTB was used to develop an enzyme-linked immunosorbent assay (ELISA) to determine the GL-3 concentrations in plasma and tissue extracts from normal individuals and patients and mice with alpha-galactosidase A deficiency (human Fabry disease). The mean (+/-1 SD) plasma GL-3 concentrations in affected male and female heterozygotes with Fabry disease were 12.6 +/- 3.7 and 1.1 +/- 0.7 microg/ml, respectively, whereas normal individuals had 0.9 +/- 0.4 microg/ml. In 5- to 6-month-old mice with alpha-galactosidase A deficiency, the average GL-3 concentrations in spleen, kidney, liver, heart, and plasma were 2790 +/- 400, 1100 +/- 93, 378 +/- 67, and 196 +/- 28 ng/mg wet wt and 5. 1 +/- 2.0 microg/ml, respectively, whereas tissues from wild-type mice contained very low or undetectable GL-3 levels. This ELISA assay should prove useful for determining the GL-3 levels, as well as for monitoring the effectiveness of therapeutic endeavors in patients with Fabry disease.

Ribosomal proteins in cell proliferation and apoptosis.

Ribosomal proteins have the complex task of coordinating protein biosynthesis to maintain cell homeostasis and survival. Recent evidence suggests that a number of ribosomal proteins have secondary functions independent of their involvement in protein biosynthesis. A number of these proteins function as cell proliferation regulators and in some instances as inducers of cell death. Specifically, expression of human ribosomal protein L13a has been shown to induce apoptosis, presumably by arresting cell growth in the G2/M phase of the cell cycle. In addition, inhibition of expression of L13a induces apoptosis in target cells, suggesting that this protein is necessary for cell survival. Similar results have been obtained in the yeast Saccharomyces cerevisiae, where inactivation of the yeast homologues of L13a, rp22 and rp23, by homologous recombination results in severe growth retardation and death. In addition, a closely related ribosomal protein, L7, arrests cells in G1 and also induces apoptosis. Thus, it appears that a group of ribosomal proteins may function as cell cycle checkpoints and compose a new family of cell proliferation regulators.

Regulation of rat hepatic 3beta-hydroxysterol delta7-reductase: substrate specificity, competitive and non-competitive inhibition, and phosphorylation/dephosphorylation.

The mechanism for the catalytic reduction of the double bond at C-7, 8 in 7-dehydrocholesterol by 3beta-hydroxysterol Delta7-reductase was investigated by testing structurally related sterols as substrates and potential inhibitors. The hepatic smooth endoplasmic reticulum was identified as the site of enzyme activity. All putative substrates contained 27 carbons, but differed from 7-dehydrocholesterol by the addition of either an ethyl substituent at C-24 (7-dehydrositosterol), a double bond at C-22 with a methyl substituent at C-24 (ergosterol), epimerization of the hydroxyl from the 3beta- to 3alpha-configuration (7-dehydroepicholesterol), or a saturated double bond at C-5,6 (lathosterol). Two non-steroidal compounds that inhibit 3beta-hydroxysterol Delta7-reductase in vivo (AY 9944 and BM 15.766) were also tested. Ergosterol, 7-dehydrositosterol, and 7-dehydroepicholesterol were reduced at C-7, 8 to form brassicasterol, sitosterol, and epicholesterol, respectively, but 75% less efficiently than 7-dehydrocholesterol. Increasing concentrations of these sterols competitively inhibited 3beta-hydroxysterol Delta7-reductase activity. The double bond at C-7,8 in lathosterol was not reduced. AY 9944 and BM 15.766 inhibited 3beta-hydroxysterol Delta7-reductase activity non-competitively. 3beta-Hydroxysterol-Delta7-reductase activity declined after microsomes were exposed to alkaline phosphatase, and enzyme activity was increased by phosphorylation with Mg2+, and ATP. These results demonstrate that the reduction of the double bond at C-7,8 requires binding of the enzyme protein with the B-ring of the sterol substrate that contains a double bond at C-5,6. The reaction is hindered by substituents located on the apolar side-chain and epimerization of the hydroxyl group in ring A to a 3alpha-configuration. 3beta-Hydroxysterol Delta7-reductase exists in two forms: an active phosphorylated form and an inactive dephosphorylated form.

Differential gene expression in apoptosis: identification of ribosomal protein 23K, a cell proliferation inhibitor.

Gene expression during the camptothecin-induced apoptotic death of human leukemic U937 cells and mouse T-cell hybridoma QW4.1 cells was studied by the mRNA differential display technique. Ten clones were confirmed to be differentially expressed, nine of which encoded novel sequences. One clone, U3.2, was induced approximately 10-fold in camptothecin-treated cells and was found to be identical to a highly basic 23-kDa human protein which contains basic leucine zipper-like motifs and has recently been identified as the human homologue of the rat ribosomal protein L13a. Northern blot analysis revealed a major mRNA of approximately 0.9 kb and a minor mRNA of approximately 1.3 kb. Overexpression of a full-length 23K cDNA, tagged with a FLAG sequence, in COS-7 cells revealed a predominantly nucleolar localization and the absence of any 23K protein from the cytoplasm. Subsequent transfection studies, using antisense phosphorothioate-modified oligonucleotides, revealed that inhibition of 23K expression results in an increased cell proliferation and greater sensitivity of U937 cells to the effects of camptothecin-induced cell death. Upregulation of 23K expression using a cDNA construct resulted in a decrease in cell proliferation and growth arrest, suggesting a role for 23K protein as a proliferation checkpoint following a cellular insult.

Murine alpha-N-acetylgalactosaminidase: isolation and expression of a full-length cDNA and genomic organization: further evidence of an alpha-galactosidase gene family.

Recent characterization of the human sequences encoding two lysosomal hydrolases, alpha-galactosidase A (alpha-Gal A) and alpha-N-acetylgalactosaminidase (alpha-GalNAc) revealed that these two enzymes with distinct enzymatic activities shared about 50% overall amino acid identity and that their genomic sequences had a conserved common gene structure. These findings suggested that these genes, which are located on different chromosomes, arose by duplication and divergence from a common ancestral gene. To further compare this alpha-galactosidase gene family, the murine alpha-GalNAc cDNA and genomic sequences were isolated and characterized. The full-length cDNA contained an open-reading frame of 1245 bp encoding a 415 amino acid polypeptide and had 5' and 3' untranslated regions of 94 and 333 bp, respectively. The coding region had 81% nucleotide and 81.9% amino acid identities with those of the corresponding human alpha-GalNAc sequence. Northern analysis revealed a single transcript of approximately 1.9 kb. The functional integrity of the cDNA was demonstrated by transient expression in COS-1 cells. The murine alpha-GalNAc genomic sequence spanned approximately 9 kb and was identical in structure with the human alpha-GalNAc gene with eight introns interrupting the coding sequence at identical positions. In addition, the deduced amino acid sequence of the murine alpha-GalNAc gene was highly homologous with alpha-GalNAc and alpha-Gal A genes from other species providing further support for a common evolutionary ancestor of the alpha-galactosidase gene family. The availability of the murine gene will permit additional evolutionary comparisons, structure/function analyses, and the generation of mice with alpha-GalNAc deficiency by gene targeting.

Human alpha-galactosidase A: characterization of the N-linked oligosaccharides on the intracellular and secreted glycoforms overexpressed by Chinese hamster ovary cells.

Human alpha-galactosidase A (alpha-Gal A) is the lysosomal glycohydrolase that cleaves the terminal alpha-galactosyl moieties of various glycoconjugates. Overexpression of the enzyme in Chinese hamster ovary (CHO) cells results in high intracellular enzyme accumulation and the selective secretion of active enzyme. Structural analysis of the N -linked oligosaccharides of the intracellular and secreted glycoforms revealed that the secreted enzyme's oligosaccharides were remarkably heterogeneous, having high mannose (63%), complex (30%), and hybrid (5%) structures. The major high mannose oligosaccharides were Man5-7GlcNAc2 species. Approximately 40% of the high mannose and 30% of the hybrid oligosaccharides had phosphate monoester groups. The complex oligosaccharides were mono-, bi-, 2,4-tri-, 2,6-tri- and tetraantennary with or without core-region fucose, many of which had incomplete outer chains. Approximately 30% of the complex oligosaccharides were mono- or disialylated. Sialic acids were mostly N -acetylneuraminic acid and occurred exclusively in alpha2, 3-linkage. In contrast, the intracellular enzyme had only small amounts of complex chains (7.7%) and had predominantly high mannose oligosaccharides (92%), mostly Man5GlcNAc2 and smaller species, of which only 3% were phosphorylated. The complex oligosaccharides were fucosylated and had the same antennary structures as the secreted enzyme. Although most had mature outer chains, none were sialylated. Thus, the overexpression of human alpha-Gal A in CHO cells resulted in different oligosaccharide structures on the secreted and intracellular glycoforms, the highly heterogeneous secreted forms presumably due to the high level expression and impaired glycosylation in the trans- Golgi network, and the predominately Man5-7GlcNAc2 cellular glycoforms resulting from carbohydrate trimming in the lysosome.

Human alpha-galactosidase A: glycosylation site 3 is essential for enzyme solubility.

Human alpha-galactosidase A (EC 3.2.1.22; alpha-Gal A) is the homodimeric glycoprotein that hydrolyses the terminal alpha-galactosyl moieties from glycolipids and glycoproteins. The type, site occupancy and function of the N-linked oligosaccharide chains on this lysosomal hydrolase were determined. Endoglycosidase treatment of the purified recombinant enzyme and mutagenesis studies indicated that three (Asn-139, Asn-192 and Asn-215) of the four potential N-glycosylation consensus sequences were occupied by complex, high-mannose and hybrid-type oligosaccharides respectively. When expressed in COS-1 cells, glycoforms with glycosylation site 1 or 2 obliterated had more than 70% of wild-type activity, and both glycoforms were secreted. In contrast, the glycoform with only site 3 eliminated had decreased activity (less than 40%); little, if any, was secreted. Expressed mutant glycoforms in which site 3 and site 1 or 2 were obliterated had little, if any, intracellular or secreted enzymic activity, and immunofluorescence microscopy revealed that the expressed mutant glycoforms were retained in the endoplasmic reticulum, presumably where they were degraded. Thus glycosylation at site 3 was crucial to the formation of soluble, active enzyme, as well as transport to the lysosome. Absence of the site 3 hybrid-type oligosaccharide exposed an adjacent, normally protected, hydrophobic region, resulting in aggregation of the enzyme polypeptide in the endoplasmic reticulum. In support of this concept, endoglycosidase H-treated enzyme or mannose-terminated enzyme expressed in Autographa californica cells also aggregated when concentrated, emphasizing that site 3 occupancy by a hybrid-type oligosaccharide was required for enzyme solubility.