Human acid sphingomyelinase structures provide insight to molecular basis of Niemann-Pick disease.

Acid sphingomyelinase (ASM) hydrolyzes sphingomyelin to ceramide and phosphocholine, essential components of myelin in neurons. Genetic alterations in ASM lead to ASM deficiency (ASMD) and have been linked to Niemann-Pick disease types A and B. Olipudase alfa, a recombinant form of human ASM, is being developed as enzyme replacement therapy to treat the non-neurological manifestations of ASMD. Here we present the human ASM holoenzyme and product bound structures encompassing all of the functional domains. The catalytic domain has a metallophosphatase fold, and two zinc ions and one reaction product phosphocholine are identified in a histidine-rich active site. The structures reveal the underlying catalytic mechanism, in which two zinc ions activate a water molecule for nucleophilic attack of the phosphodiester bond. Docking of sphingomyelin provides a model that allows insight into the selectivity of the enzyme and how the ASM domains collaborate to complete hydrolysis. Mapping of known mutations provides a basic understanding on correlations between enzyme dysfunction and phenotypes observed in ASMD patients.

Impact of cysteine variants on the structure, activity, and stability of recombinant human α-galactosidase A.

Recombinant human α-galactosidase A (rhαGal) is a homodimeric glycoprotein deficient in Fabry disease, a lysosomal storage disorder. In this study, each cysteine residue in rhαGal was replaced with serine to understand the role each cysteine plays in the enzyme structure, function, and stability. Conditioned media from transfected HEK293 cells were assayed for rhαGal expression and enzymatic activity. Activity was only detected in the wild type control and in mutants substituting the free cysteine residues (C90S, C174S, and the C90S/C174S). Cysteine-to-serine substitutions at the other sites lead to the loss of expression and/or activity, consistent with their involvement in the disulfide bonds found in the crystal structure. Purification and further characterization confirmed that the C90S, C174S, and the C90S/C174S mutants are enzymatically active, structurally intact and thermodynamically stable as measured by circular dichroism and thermal denaturation. The purified inactive C142S mutant appeared to have lost part of its alpha-helix secondary structure and had a lower apparent melting temperature. Saturation mutagenesis study on Cys90 and Cys174 resulted in partial loss of activity for Cys174 mutants but multiple mutants at Cys90 with up to 87% higher enzymatic activity (C90T) compared to wild type, suggesting that the two free cysteines play differential roles and that the activity of the enzyme can be modulated by side chain interactions of the free Cys residues. These results enhanced our understanding of rhαGal structure and function, particularly the critical roles that cysteines play in structure, stability, and enzymatic activity.

Structures of a pan-specific antagonist antibody complexed to different isoforms of TGFß reveal structural plasticity of antibody-antigen interactions.

Various important biological pathways are modulated by TGFß isoforms; as such they are potential targets for therapeutic intervention. Fresolimumab, also known as GC1008, is a pan-TGFß neutralizing antibody that has been tested clinically for several indications including an ongoing trial for focal segmental glomerulosclerosis. The structure of the antigen-binding fragment of fresolimumab (GC1008 Fab) in complex with TGFß3 has been reported previously, but the structural capacity of fresolimumab to accommodate tight interactions with TGFß1 and TGFß2 was insufficiently understood. We report the crystal structure of the single-chain variable fragment of fresolimumab (GC1008 scFv) in complex with target TGFß1 to a resolution of 3.00 Å and the crystal structure of GC1008 Fab in complex with TGFß2 to 2.83 Å. The structures provide further insight into the details of TGFß recognition by fresolimumab, give a clear indication of the determinants of fresolimumab pan-specificity and provide potential starting points for the development of isoform-specific antibodies using a fresolimumab scaffold.

Identification and quantitation of Vesivirus 2117 particles in bioreactor fluids from infected Chinese hamster ovary cell cultures.

The prevention of adventitious agent contamination is a top priority throughout the entire biopharmaceutical production process. For example, although viral contamination of cell banks or cell cultures is rare, it can result in serious consequences (e.g., shutdown and decontamination of manufacturing facilities). To ensure virus free production, numerous in vivo and in vitro adventitious agent assays and biophysical characterizations such as electron microscopy are conducted on cell banks, raw materials, process materials, and drug substances throughout the manufacturing process. Molecular assays such as PCR and other nucleotide-based techniques are also routinely used for screening and identification of any viral agents. However, modern techniques in protein identification of complex protein mixtures have not yet been effectively integrated throughout the industry into current viral testing strategies. Here, we report the identification and quantitation of Vesivirus 2117 particles in bioreactor fluid from infected Chinese hamster ovary cell cultures by global protein sequencing using mass spectrometry in combination with multi-dimensional liquid-chromatography. Following mass spectrometric data acquisition and rigorous data analysis, six virus specific peptides were identified. These peptides were fragments of two structural proteins, capsid protein pre-cursor (four unique peptides) and small structural protein (two unique peptides), from the same species: Vesivirus 2117. Using stable heavy isotope-labeled peptides as internal standards, we also determined the absolute concentration of Vesivirus particles in the bioreactor fluid and the ratio of two capsid proteins (VP1:VP2) in the particles as approximately 9:1. The positive identification of Vesivirus 2117 was subsequently confirmed by RT-PCR.

Characterization of the N370S mutant of glucocerebrosidase by hydrogen/deuterium exchange mass spectrometry.

An asparagine-to-serine substitution at residue 370 (N370S) in glucocerebrosidase (GCase) is the most prevalent mutation leading to Gaucher's disease, the most common lysosomal storage disorder. Two types of hydrogen/deuterium exchange experiment coupled with proteolysis and liquid chromatography-mass spectrometry (HDX-MS) were used to investigate the dynamic properties and unfolding stability of wt, R495H, and N370S GCases in the presence and absence of ligands. R495H GCase is used for enzyme replacement therapy and is considered to be a wt surrogate, whereas N370S is the most prevalent mutation leading to Gaucher's disease. Time-course HDX experiments of the GCases were performed under near-physiological conditions to detect the protein's local unfolding motions at a submolecular level. In guanidine-titration experiments, HDX reactions were performed with various concentrations of a chemical denaturant to provide the global stability of the proteins. The two types of experiment showed that all three purified GCases, wt, R495H, and N370S, have virtually identical local unfolding motions and global stabilities in solution. Combined with previous X-ray crystallographic studies, which showed indistinguishable backbone conformations for N370S and R495H GCase mutants and very similar melting temperatures for the wt, R495H, and N370S mutants, all three GCases are likely to have virtually identical structural and dynamic properties in solution. The guanidine-titration experiments revealed that the pharmacological chaperone, isofagomine (IFG), interacts more weakly with the N370S mutant than with the R495H GCase; this is consistent with the higher IC(50) value of IFG against N370S than against R495H. The time-course experiments showed that IFG restricts the local unfolding motions of N370S in the same way as those of R495H when the ligand saturates the proteins.

Strategies for Neoglycan conjugation to human acid α-glucosidase.

Engineering proteins for selective tissue targeting can improve therapeutic efficacy and reduce undesired side effects. The relatively high dose of recombinant human acid α-glucosidase (rhGAA) required for enzyme replacement therapy of Pompe disease may be attributed to less than optimal muscle uptake via the cation-independent mannose 6-phosphate receptor (CI-MPR). To improve muscle targeting, Zhu et al. (1) conjugated periodate oxidized rhGAA with bis mannose 6-phosphate bearing synthetic glycans and achieved 5-fold greater potency in a murine Pompe efficacy model. In the current study, we systematically evaluated multiple strategies for conjugation based on a structural homology model of GAA. Glycan derivatives containing succinimide, hydrazide, and aminooxy linkers targeting free cysteine, lysines, and N-linked glycosylation sites on rhGAA were prepared and evaluated in vitro and in vivo. A novel conjugation method using enzymatic oxidation was developed to eliminate side oxidation of methionine. Conjugates derived from periodate oxidized rhGAA still displayed the greatest potency in the murine Pompe model. The efficiency of conjugation and its effect on catalytic activity were consistent with predictions based on the structural model and supported its use in guiding selection of appropriate chemistries.

X-ray and biochemical analysis of N370S mutant human acid ß-glucosidase.

Gaucher disease is caused by mutations in the enzyme acid ß-glucosidase (GCase), the most common of which is the substitution of serine for asparagine at residue 370 (N370S). To characterize the nature of this mutation, we expressed human N370S GCase in insect cells and compared the x-ray structure and biochemical properties of the purified protein with that of the recombinant human GCase (imiglucerase, Cerezyme®). The x-ray structure of N370S mutant acid ß-glucosidase at acidic and neutral pH values indicates that the overall folding of the N370S mutant is identical to that of recombinant GCase. Subtle differences were observed in the conformation of a flexible loop at the active site and in the hydrogen bonding ability of aromatic residues on this loop with residue 370 and the catalytic residues Glu-235 and Glu-340. Circular dichroism spectroscopy showed a pH-dependent change in the environment of tryptophan residues in imiglucerase that is absent in N370S GCase. The mutant protein was catalytically deficient with reduced V(max) and increased K(m) values for the substrate p-nitrophenyl-ß-D-glucopyranoside and reduced sensitivity to competitive inhibitors. N370S GCase was more stable to thermal denaturation and had an increased lysosomal half-life compared with imiglucerase following uptake into macrophages. The competitive inhibitor N-(n-nonyl)deoxynojirimycin increased lysosomal levels of both N370S and imiglucerase 2-3-fold by reducing lysosomal degradation. Overall, these data indicate that the N370S mutation results in a normally folded but less flexible protein with reduced catalytic activity compared with imiglucerase.

Use of direct fluorescence labeling and confocal microscopy to determine the biodistribution of two protein therapeutics, Cerezyme and Ceredase.

Efficient targeting of therapeutic reagents to tissues and cell types of interest is critical to achieving therapeutic efficacy and avoiding unwanted side effects due to offtarget uptake. To increase assay efficiency and reduce the number of animals used per experiment during preclinical development, we used a combination of direct fluorescence labeling and confocal microscopy to simultaneously examine the biodistribution of two therapeutic proteins, Cerezyme and Ceredase, in the same animals. We show that the fluorescent tags do not interfere with protein uptake and localization. We are able to detect Cerezyme and Ceredase in intact cells and organs and demonstrate colocalization within target cells using confocal microscopy. In addition, the relative amount of protein internalized by different cell types can be quantified using cell type-specific markers and morphometric analysis. This approach provides an easy and straightforward means of assessing the tissue and cell type-specific biodistribution of multiple protein therapeutics in target organs using a minimal number of animals.

A multi-tiered analytical approach for the analysis and quantitation of high-molecular-weight aggregates in a recombinant therapeutic glycoprotein.

In this study, we have investigated sedimentation velocity ultracentrifugation (AUC-SV), size exclusion chromatography (SEC), and circular dichroism (CD) methods for the detection and quantitation of protein aggregates using recombinant acid alpha-glucosidase (rhGAA) as a model. The results of this study showed that the formation and molecular weight distribution of rhGAA aggregated species were dependent upon the formulation conditions as well as the storage or stress conditions used to induce aggregation. The utility of CD as a probe for non-native, aggregated species was affirmed, as this method was sensitive to rhGAA aggregation levels of < or = 4%. An extensive evaluation of AUC-SV variability was performed using nine levels of spiked rhGAA aggregate that were analyzed on six occasions. Based on our data, the precision of the AUC-SV results increased with increasing levels of aggregate, with a mean RSD of 37.2%. The limit of quantitation (LOQ) for the AUC-SV method, which was based on a Precision criterion of RSD < 20%, was determined to be > or = 3% aggregated rhGAA. The Precision and LOQ of the SEC method, determined using the same rhGAA sample set, was found to be 3.8% and > or = 0.2%, respectively. In general, there was good agreement between the levels of aggregated rhGAA determined using the AUC-SV and SEC methods, with a slight positive bias noted for the AUC-SV results. These studies emphasize the value of applying multiple, well-characterized analytical tools in the evaluation of therapeutic protein aggregation.

In vitro and in vivo evaluation of a non-carbohydrate targeting platform for lysosomal proteins.

Lysosomal storage diseases arise from a genetic loss-of-function defect in enzymes mediating key catabolic steps resulting in accumulation of substrate within the lysosome. Treatment of several of these disorders has been achieved by enzyme replacement therapy (ERT), in which a recombinant version of the defective enzyme is expressed in vitro and administered by infusion. However, in many cases the biodistribution of the administered protein does not match that of the accumulated substrate due to the glycosylation-mediated clearance of the enzymes from circulation, resulting in poor or absent substrate clearance from some tissues. To overcome this limitation, we have evaluated several peptide-based targeting motifs to redirect recombinant human alpha-galactosidase (rhalphaGal) to specific receptors. A reversible thiol-based PEGylation chemistry was developed to achieve multivalent peptide display with lysosomal release. In vitro, cell uptake was peptide dependent and independent of the normal mannose-6-phosphate receptor mediated pathway. Surprisingly, despite increased plasma half-life and decreased liver uptake, none of the peptide conjugates showed significantly altered biodistribution in alphaGal-knockout mice. This suggests that these peptide-based targeting motifs are unlikely to provide substantial therapeutic benefit likely due to the complexity of factors affecting PK and biodistribution.

Binding parameters and thermodynamics of the interaction of imino sugars with a recombinant human acid alpha-glucosidase (alglucosidase alfa): insight into the complex formation mechanism.

BACKGROUND: Recently, enzyme enhancement therapy (EET) for Pompe disease involving imino sugars, which act as potential inhibitors of acid alpha-glucosidases in vitro, to improve the stability and/or transportation of mutant acid alpha-glucosidases in cells was studied and attracted interest. However, the mechanism underlying the molecular interaction between the imino sugars and the enzyme has not been clarified yet. METHODS: We examined the inhibitory and binding effects of four imino sugars on a recombinant human acid alpha-glucosidase, alglucosidase alfa, by means of inhibition assaying and isothermal titration calorimetry (ITC). Furthermore, we built structural models of complexes of the catalytic domain of the enzyme with the imino sugars bound to its active site by homology modeling, and examined the molecular interaction between them. RESULTS: All of the imino sugars examined exhibited a competitive inhibitory action against the enzyme, 1-deoxynojirimycin (DNJ) exhibiting the strongest action among them. ITC revealed that one compound molecule binds to one enzyme molecule and that DNJ most strongly binds to the enzyme among them. Structural analysis revealed that the active site of the enzyme is almost completely occupied by DNJ. CONCLUSION: These biochemical and structural analyses increased our understanding of the molecular interaction between a human acid alpha-glucosidase and imino sugars.

Development of a simple and rapid method for producing non-fucosylated oligomannose containing antibodies with increased effector function.

Glycosylation in the Fc region of antibodies has been shown to play an important role in antibody function. In the current study, glycosylation of human monoclonal antibodies was metabolically modulated using a potent alpha-mannosidase I inhibitor, kifunensine, resulting in the production of antibodies with oligomannose-type N-glycans. Growing Chinese hamster ovary cells for 11 days in batch culture with a single treatment of kifunensine was sufficient to elicit this effect without any significant impact on cell viability or antibody production. Antibodies expressed in the presence of kifunensine at a concentration as low as 60 ng/mL contained mainly oligomannose-type glycans and demonstrated increased ADCC activity and affinity for FcgammaRIIIA, but reduced C1q binding. Although the kifunensine-mediated shift to oligomannose-type glycans could, in theory, result in rapid clearance of the antibody through increased mannose receptor binding, the serum levels of antibody in mice were not significantly altered up to 168 h following injection. The use of kifunensine provides a simple and rapid method for the production of antibodies with increased ADCC without the time-consuming need to re-engineer either the antibody molecule or the host cell line.

LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of beta-glucocerebrosidase.

beta-glucocerebrosidase, the enzyme defective in Gaucher disease, is targeted to the lysosome independently of the mannose-6-phosphate receptor. Affinity-chromatography experiments revealed that the lysosomal integral membrane protein LIMP-2 is a specific binding partner of beta-glucocerebrosidase. This interaction involves a coiled-coil domain within the lumenal domain. beta-glucocerebrosidase activity and protein levels were severely decreased in LIMP-2-deficient mouse tissues. Analysis of fibroblasts and macrophages isolated from these mice indicated that the majority of beta-glucocerebrosidase was secreted. Missorting of beta-glucocerebrosidase was also evident in vivo, as protein and activity levels were significantly higher in sera from LIMP-2-deficient mice compared to wild-type. Reconstitution of LIMP-2 in LIMP-2-deficient fibroblasts led to a rescue of beta-glucocerebrosidase levels and distribution. LIMP-2 expression also led to lysosomal transport of a beta-glucocerebrosidase endoplasmic reticulum retention mutant. These data support a role for LIMP-2 as the mannose-6-phosphate-independent trafficking receptor for beta-glucocerebrosidase.

Structural and biochemical studies on Pompe disease and a "pseudodeficiency of acid alpha-glucosidase".

We constructed structural models of the catalytic domain and the surrounding region of human wild-type acid alpha-glucosidase and the enzyme with amino acid substitutions by means of homology modeling, and examined whether the amino acid replacements caused structural and biochemical changes in the enzyme proteins. Missense mutations including p.R600C, p.S619R and p.R437C are predicted to cause apparent structural changes. Nonsense mutation of p.C103X terminates the translation of acid alpha-glucosidase halfway through its biosynthesis and is deduced not to allow formation of the active site pocket. The mutant proteins resulting from these missense and nonsense mutations found in patients with Pompe disease are predictably unstable and degraded quickly in cells. The structural change caused by p.G576S is predicted to be small, and cells from a subject homozygous for this amino acid substitution exhibited 15 and 11% of the normal enzyme activity levels for an artificial substrate and glycogen, respectively, and corresponding amounts of the enzyme protein on Western blotting. No accumulation of glycogen was found in organs including skeletal muscle in the subject, and thus the residual enzyme activity could protect cells from glycogen storage. On the other hand, p.E689K, which is known as a neutral polymorphism, little affected the three-dimensional structure of acid alpha-glucosidase. Structural study on a mutant acid alpha-glucosidase in silico combined with biochemical investigation is useful for understanding the molecular pathology of Pompe disease.

N-glycans of recombinant human acid alpha-glucosidase expressed in the milk of transgenic rabbits.

Pompe disease is a lysosomal glycogen storage disorder characterized by acid alpha-glucosidase (GAA) deficiency. More than 110 different pathogenic mutations in the gene encoding GAA have been observed. Patients with this disease are being treated by intravenous injection of recombinant forms of the enzyme. Focusing on recombinant approaches to produce the enzyme means that specific attention has to be paid to the generated glycosylation patterns. Here, human GAA was expressed in the mammary gland of transgenic rabbits. The N-linked glycans of recombinant human GAA (rhAGLU), isolated from the rabbit milk, were released by peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase F. The N-glycan pool was fractionated and purified into individual components by a combination of anion-exchange, normal-phase, and Sambucus nigra agglutinin-affinity chromatography. The structures of the components were analyzed by 500 MHz one-dimensional and 600 MHz cryo two-dimensional (total correlation spectroscopy [TOCSY] nuclear Overhauser enhancement spectroscopy) (1)H nuclear magnetic resonance spectroscopy, combined with two-dimensional (31)P-filtered (1)H-(1)H TOCSY spectroscopy, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and high-performance liquid chromatography (HPLC)-profiling of 2-aminobenzamide-labeled glycans combined with exoglycosidase digestions. The recombinant rabbit glycoprotein contained a broad array of different N-glycans, comprising oligomannose-, hybrid-, and complex-type structures. Part of the oligomannose-type glycans showed the presence of phospho-diester-bridged N-acetylglucosamine. For the complex-type glycans (partially) (alpha2-6)-sialylated (nearly only N-acetylneuraminic acid) diantennary structures were found; part of the structures were (alpha1-6)-core-fucosylated or (alpha1-3)-fucosylated in the upper antenna (Lewis x). Using HPLC-mass spectrometry of glycopeptides, information was generated with respect to the site-specific location of the various glycans.

Effect of mannose chain length on targeting of glucocerebrosidase for enzyme replacement therapy of Gaucher disease.

Recombinant human glucocerebrosidase (imiglucerase, Cerezyme) is used in enzyme replacement therapy for Gaucher disease. Complex oligosaccharides present on Chinese hamster ovary cell-expressed glucocerebrosidase (GCase) are enzymatically remodeled into a mannose core, facilitating mannose receptor-mediated uptake into macrophages. Alternative expression systems could be used to produce GCase containing larger oligomannose structures, offering the possibility of an improvement in targeting to macrophages. A secondary advantage of these expression systems would be to eliminate the need for carbohydrate remodeling. Here, multiple expression systems were used to produce GCase containing primarily terminal oligomannose, from Man2 to Man9. GCase from these multiple expression systems was compared to Cerezyme with respect to affinity for mannose receptor and serum mannose-binding lectin (MBL), macrophage uptake, and intracellular half-life. In vivo studies comparing clearance and targeting of Cerezyme and the Man9 form of GCase were carried out in a Gaucher mouse model (D409V/null). Mannose receptor binding, macrophage uptake, and in vivo targeting were similar for all forms of GCase. Increased MBL binding was observed for all forms of GCase having larger mannose structures than those of Cerezyme, which could influence pharmacokinetic behavior. These studies demonstrate that although alternative cell expression systems are effective for producing oligomannose-terminated glucocerebrosidase, there is no biochemical or pharmacological advantage in producing GCase with an increased number of mannose residues. The display of alternative carbohydrate structures on GCase expressed in these systems also runs the risk of undesirable consequences, such as an increase in MBL binding or a possible increase in immunogenicity due to the presentation of non-mammalian glycans.

Chemical chaperones and permissive temperatures alter localization of Gaucher disease associated glucocerebrosidase variants.

Point mutations in the lysosomal hydrolase, glucocerebrosidase (GC), can cause Gaucher disease, a common lysosomal storage disease. Several clinically important GC mutations impede folding in the endoplasmic reticulum (ER) and target these enzymes for ER-associated degradation (ERAD). The removal of these misfolded proteins decreases the lysosomal concentration of GC, which results in glucosylceramide accumulation. The most common GC variant, N370S, and other clinically relevant variants, G202R and L444P, exhibit different cellular localization patterns in patient-derived fibroblasts. We show that these distributions can be altered by manipulation of the ER folding environment, either by chemical chaperones or by temperature shifts. N370S, L444P, and G202R GC are destabilized in the neutral pH environment of the ER, rendering them prone to ERAD. Fibroblasts harboring the G202R and L444P GC mutations grown at 30 degrees C localize the mutant proteins to the lysosome, and this increases total GC activity. Both of these temperature-sensitive mutants appear to be stable at 37 degrees C once they are trafficked to the low pH environment of the lysosome. Chemical chaperones correct the ER instability and significant ER retention of G202R GC. N370S is also destabilized under ER simulating conditions, a deficiency that is corrected by chemical chaperone binding. These data clearly show manipulating the ER environment with chemical chaperones increases the lysosomal concentration of partially active GC variants and suggest that small molecules could be used to treat Gaucher disease.

Effect of genetic background on glycosylation heterogeneity in human antithrombin produced in the mammary gland of transgenic goats.

Glycosylation is involved in the correct folding, targeting, bioactivity and clearance of therapeutic glycoproteins. With the development of transgenic animals as expression systems it is important to understand the impact of different genetic backgrounds and lactations on glycosylation. We have evaluated the glycosylation of recombinant antithrombin produced in several transgenic goat lines, from cloned animals and from different types of lactation including induced lactations. Our results show glycosylation patterns from the protein expressed in animals, derived from the same founder goat, are mostly comparable. Furthermore, the protein expressed in two cloned goats had highly consistent oligosaccharide profiles and similar carbohydrate composition. However, there were significantly different oligosaccharide profiles from the proteins derived from different founder goats. Artificial induction of lactation did not have significant effects on overall carbohydrate structures when compared to natural lactation. The only major difference was that recombinant antithrombin from induced lactations contained a slightly higher ratio of N-acetylneuraminic acid to N-glycolylneuraminic acid and less amount of oligosaccharides containing N-glycolylneuraminic acid. The oligosaccharides from all animals were a mixture of high mannose-, hybrid- and complex-type oligosaccharides. Sialic acid was present as alpha-2,6-linkage and no alpha-1,3-linked galactose was observed. These results indicate that transgenic animals with closely related genetic backgrounds express recombinant protein with comparable glycosylation.

Lysosomal acid alpha-glucosidase consists of four different peptides processed from a single chain precursor.

Pompe's disease is caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). GAA is synthesized as a 110-kDa precursor containing N-linked carbohydrates modified with mannose 6-phosphate groups. Following trafficking to the lysosome, presumably via the mannose 6-phosphate receptor, the 110-kDa precursor undergoes a series of complex proteolytic and N-glycan processing events, yielding major species of 76 and 70 kDa. During a detailed characterization of human placental and recombinant human GAA, we found that the peptides released during proteolytic processing remained tightly associated with the major species. The 76-kDa form (amino acids (aa) 122-782) of GAA is associated with peptides of 3.9 kDa (aa 78-113) and 19.4 kDa (aa 792-952). The 70-kDa form (aa 204-782) contains the 3.9- and 19.4-kDa peptide species as well as a 10.3-kDa species (aa 122-199). A similar set of proteolytic fragments has been identified in hamster GAA, suggesting that the multicomponent character is a general phenomenon. Rabbit anti-peptide antibodies have been generated against sequences in the proteolytic fragments and used to demonstrate the time course of uptake and processing of the recombinant GAA precursor in Pompe's disease fibroblasts. The results indicate that the observed fragments are produced intracellularly in the lysosome and not as a result of nonspecific proteolysis during purification. These data demonstrate that the mature forms of GAA characterized by polypeptides of 76 or 70 kDa are in fact larger molecular mass multicomponent enzyme complexes.

N-linked oligosaccharide analysis of glycoprotein bands from isoelectric focusing gels.

Glycoproteins often display a complex isoelectric focusing profile because of the presence of negatively charged carbohydrates, such as sialic acid, phosphorylated mannose, and sulfated GalNAc. Until now, understanding the role of these charged carbohydrates in determining the isoelectric focusing profile has been limited to observing pattern shifts following complete removal of the sugars in question. We have developed a simple and sensitive method for analyzing N-linked oligosaccharides from the individual isoelectric focusing bands of a glycoprotein using recombinant human thyroid-stimulating hormone as a model system. N-linked oligosaccharides were released and profiled from individual bands following electroblotting of isoelectric focusing gels. As might be predicted, high-pH anion-exchange chromatography-pulsed amperometric detection and matrix-assisted laser desorption/ionization-time of flight analyses indicated that the bands that migrated closer to the positive electrode contained more sialylated N-linked oligosaccharides. The sialic acid content of these bands correlated with that predicted from the corresponding oligosaccharide analyses.