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Biomedical subjects

D A Brooks

Publications and source records attributed to D A Brooks.

At least 19 recordsLinked to original sources
PubMed1999

Processing of normal lysosomal and mutant N-acetylgalactosamine 4-sulphatase: BiP (immunoglobulin heavy-chain binding protein) may interact with critical protein contact sites.

The lysosomal hydrolase N-acetylgalactosamine-4-sulphatase (4-sulphatase) is essential for the sequential degradation of the glycosaminoglycans, dermatan and chondroitin sulphate and, when deficient, causes the lysosomal storage disorder mucopolysaccharidosis type VI. The cysteine at codon 91 of human 4-sulphatase was identified previously as a key residue in the active site of the enzyme and was mutated by site-directed mutagenesis to produce a 4-sulphatase in which cysteine-91 was replaced by a threonine residue (C91T). The C91T mutation caused a loss of 4-sulphatase activity, a detectable protein conformational change and a lower level of intracellular 4-sulphatase protein [Brooks, Robertson, Bindloss, Litjens, Anson, Peters, Morris and Hopwood (1995) Biochem. J. 307, 457-463]. In the present study, we report that C91T is synthesized normally in the endoplasmic reticulum as a 66 kDa glycosylated protein, which is very similar in size to wild-type 4-sulphatase. However, C91T neither underwent normal Golgi processing, shown by lack of modification to form mannose 6-phosphate residues on its oligosaccharide side chains, nor did it traffic to the lysosome to undergo normal endosomal-lysosomal proteolytic processing. Instead, C91T remained in an early biosynthetic compartment and was degraded. The molecular chaperone, immunoglobulin binding protein (BiP), was associated with newly-synthesized wild-type and mutant 4-sulphatase proteins for extended periods, but no direct evidence was found for involvement of BiP in the retention or degradation of the C91T protein. This suggested that prolonged association of mutant protein with BiP does not necessarily infer involvement of BiP in the quality control process, as previously implied in the literature. The predicted BiP binding sites on 4-sulphatase map to beta-strands and alpha-helices, which are co-ordinated together in the folded molecule, indicating that BiP interacts with critical protein folding or contact sites on 4-sulphatase.

Binding Sites↗
PubMed1999

Altered trafficking and turnover of LAMP-1 in Pompe disease-affected cells.

The lysosome-associated membrane protein (LAMP-1) is elevated in the cells and plasma from lysosomal storage disorder-affected individuals; however, the mechanism of this elevation is not well defined. In this study we have investigated the synthesis, glycoprocessing, trafficking, and turnover of LAMP-1 in human skin fibroblasts from Pompe disease patients and control individuals. There were similar levels of LAMP-1 synthesis in both cell types, but glycoprocessing was retarded in Pompe (T1/2 = 25 min) compared to control (T1/2 = 17 min) fibroblasts. There was also a marked delay in trafficking of LAMP-1 to lysosomes of Pompe (T1/2 = 200 min) compared to control (T1/2 = 100 min) cells. A proportion of newly synthesized LAMP-1 (5.4% in Pompe and 8.5% in controls) was trafficked out of the cell (T1/2 = 3.5 h in controls) and, although significantly smaller than the lysosomal form, still had a transmembrane domain and cytoplasmic tail. In contrast, a soluble lysosomal pool of LAMP-1 had no tail sequence, suggesting that it had been clipped from the membrane. In turnover studies, LAMP-1 was more stable in Pompe (T1/2 = 4.9 days) compared to control (T1/2 = 1. 6 days) cells, implying either reduced proteolysis or lysosomal function, in Pompe cells. These results indicate altered traffic and turnover of LAMP-1 in storage disorders and identify different intracellular and extracellular pools of soluble LAMP-1, suggesting alternative trafficking pathways.

Amino Acid Sequence↗
PubMed1999

Immune response to enzyme replacement therapy: 4-sulfatase epitope reactivity of plasma antibodies from MPS VI cats.

The mucopolysaccharidoses (MPS) are a group of multiple pathology disorders which are part of a larger group of genetic diseases known as lysosomal storage disorders. Enzyme replacement therapy (ERT) has been developed as a therapy for MPS patients. However, immune responses to ERT have been reported in MPS animal models and in human Gaucher patients. Antibodies can have adverse effects during ERT, which include hypersensitivity/anaphylactic reactions, enzyme inactivation, and enzyme degradation. This study aimed to characterize the immune response to ERT in a feline model of MPS VI, by defining the epitope reactivity of cat plasma antibody against human recombinant N-acetylgalactosamine 4-sulfatase (4-sulfatase) replacement protein. For MPS VI cat plasma, antibody reactivity was observed prior to ERT, with distinct regions of 4-sulfatase linear sequence displaying low affinity antibody reactivity. There was an increase in antibody titer to 4-sulfatase for MPS VI cats post-ERT, with the majority of the immune response detected to linear sequence epitopes. One cat displayed a high titer and high affinity epitope reactivity following prolonged exposure (>/=9 months) to the replacement protein. MPS VI cats on shorter term ERT (3 months) showed high titers to 4-sulfatase and similar patterns of epitope reactivity, but lower affinity antibody reactivity, when compared to the latter cat. This study reports the linear amino acid sequence reactivity and nature of the immune response produced to 4-sulfatase before and after ERT. The monitoring of antibody production during replacement therapy is an important consideration for patient management, as high titer antibodies can affect the efficacy of therapy.

Animals↗
PubMed1999

Immune response to enzyme replacement therapy in lysosomal storage disorder patients and animal models.

The lysosomal storage disorders (LSD) are a group of severe multiple pathology disorders characterized by enzyme deficiencies which cause the lysosomal accumulation of undegraded or partially degraded macromolecules. Enzyme replacement therapy (ERT) has been developed as a therapy for LSD patients. However, immune responses to ERT have been reported in some individuals from LSD animal model and LSD human patient studies. Antibodies can have adverse effects during ERT, which include hypersensitivity/anaphylactic reactions, enzyme inactivation, altered targeting, and increased enzyme turnover. The monitoring of antibody production during replacement therapy is an important consideration for patient management, as high-titer antibodies can affect the safety and efficacy of the therapy.

Animals↗
PubMed1999

Autologous transplantation of retrovirally transduced bone marrow or neonatal blood cells into cats can lead to long-term engraftment in the absence of myeloablation.

Autologous transplantation of retrovirally transduced bone marrow (BM) or neonatal blood cells was carried out on eight cats (ranging in age from 2 weeks to 12 months) with mucopolysaccharidosis type VI (MPS VI). The transducing vector contained the full-length cDNA encoding human arylsulfatase B (hASB), the enzymatic activity deficient in this lysosomal storage disorder. Following transplantation, the persistence of transduced cells and enzymatic expression were monitored for more than 2 years. Five of the cats received no myeloablative preconditioning, two cats received 370-390 cGy of total body irradiation (TBI), and one cat received 190 cGy TBI. Evidence of transduced cells, as judged by enzymatic activity and PCR detection of the provirus, was demonstrated in granulocytes, lymphocytes, or BM cells of the treated animals up to 31 months after transplantation. Radiation preconditioning was not required to achieve these results, nor were they dependent on the recipient's age. However, despite the long-term persistence of transduced cells, the levels of ASB activity in the transplanted animals was low, and no clinical improvements were detected. These data provide evidence for the long-term persistence of retrovirally transduced feline hematopoietic cells, and further documentation that engraftment of transduced cells can be achieved in the absence of myeloablation. Consistent with previous bone marrow transplantation studies, these results also suggest that to achieve clinical improvement of MPS VI, particularly in the skeletal system, high-level expression of ASB must be achieved in the treated animals and improved techniques for targeting the expressed enzyme to specific sites of pathology (e.g. chondrocytes) must be developed.

Animals↗
PubMed1998

Genotype-phenotype correlations in mucopolysaccharidosis type I using enzyme kinetics, immunoquantification and in vitro turnover studies.

Fibroblasts from 16 patients with known alpha-L-iduronidase gene mutations and different clinical phenotypes of mucopolysaccharidosis type I (MPS I) were investigated in order to establish genotype/phenotype correlations. Enzyme kinetic studies were performed using the specific alpha-L-iduronidase substrate iduronosyl anhydro[1-3H]mannitol-6-sulfate. Specific residual enzyme activities were estimated using the kinetic parameters and an immunoquantification assay which determines levels of alpha-L-iduronidase protein. Cells were cultured in the presence of [35S]sulfate and the in vivo degradation of accumulated labelled glycosaminoglycans measured after different chase times. Residual enzyme activity and different amounts of residual enzyme protein were present in extracts from 9 of 16 cell lines covering a wide spectrum of clinical severity. Catalytic capacity, calculated as the product of kcat/Km and ng iduronidase protein per mg cell protein, was shown in most cases to be directly related to the severity of clinical phenotype, with up to 7% of normal values for patients with the attenuated form of MPS I (Scheie) and less than 0.13% for severely affected patients (Hurler) In vitro turnover studies allowed further refinement of correlations between genotype and phenotype. Scheie disease compared to Hurler disease patients were shown to accumulate smaller amounts of glycosaminoglycans that were also turned over faster. A combination of turnover and residual enzyme data established a correlation between the genotype, the biochemical phenotype and the clinical course of this lysosomal storage disorder.

Cell Line↗
PubMed1998

Immune response to enzyme replacement therapy: clinical signs of hypersensitivity reactions and altered enzyme distribution in a high titre rat model.

Immune responses to enzyme replacement therapy (ERT) have been reported and can result in a hypersensitivity/anaphylactic reaction during or immediately after enzyme infusion. We have investigated the infusion of the lysosomal enzyme N-acetylgalactosamine 4-sulphatase (4-sulphatase) into immunized, high titre rats as a model of immune response to ERT. To simulate ERT, high and low titre rats were infused with different doses of radiolabelled recombinant human 4-sulphatase (3H-rh4S). There was evidence of altered targeting, inactivation and degradation of 4-sulphatase in high titre (titre 1024000) compared to low titre (titre 64) rats. There was more 4-sulphatase enzyme activity detected in 5 mg/kg high titre rats when compared to 1 mg/kg high titre rats, suggesting that the antibodies could be saturable in vivo. However, the rats treated with 5 mg/kg 3H-rh4S all had clinical signs of hypersensitivity reactions to 4-sulphatase infusion. There were no apparent signs of adverse reactions in either the high titre 1 mg/kg rats or the low titre rats (1, 5 mg/kg). The high titre 5 mg/kg rats also had changes in 3H-rh4S distribution, with lower levels delivered to the liver and a marked increase in the level remaining in plasma, when compared to either 1 mg/kg high titre rats or low titre rats (1, 5 mg/kg).

Anaphylaxis↗
PubMed1997

Enzyme replacement therapy in Mucopolysaccharidosis VI: evidence for immune responses and altered efficacy of treatment in animal models.

Enzyme replacement therapy (ERT) can potentially result in an immunological response to the introduced protein. The immunological response by Mucopolysaccharidosis type VI (MPS VI) cats to recombinant human N-acetylgalactosamine 4-sulfatase (rh4S) ERT has been investigated. Plasma antibody titres to rh4S were detected in untreated MPS VI and normal control cats, but the antibody titres to rh4S were higher in ERT treated MPS VI cats. The reactivity by cats to rh4S did not appear to be just due to species cross reactivity, as plasma antibodies from normal control, MPS VI and MPS VI ERT cats reacted equally with feline and human 4-sulfatase. Normal control and MPS VI human plasma also had antibody titres to rh4S. Plasma antibodies to rh4S, from an ERT treated cat, could be temporarily removed from circulation by enzyme infusion, confirming specificity for rh4S and indicating a possible window for ERT in the absence of antibody. In enzyme distribution studies with 3H-rh4S, evidence of altered targeting, and enzyme inactivation and degradation were observed in high compared to low titre rats. In high titre rats, the observed loss of 3H-label from vacuolar organelles of the liver may represent either degradation of antibody bound 3H-rh4S for reutilisation within the liver, or antigen presentation. The development of high titre antibody may have a detrimental effect on the efficacy of ERT.

Animals↗
PubMed1997

A membrane protein primarily associated with the lysosomal compartment.

A monoclonal antibody designated MBR 39 has been generated against a membrane associated protein found selectively on lysosomes. MBR 39 reacts with the cytosolic face of the lysosome and was used to develop an organelle binding assay which reacted with high density organelles characteristic of lysosomes. These organelles contained lysosomal enzyme markers which included the integral membrane protein acetyl-CoA:alpha-glucosaminide N-acetyltransferase and the soluble lysosomal enzyme markers acid phosphatase (mature form), beta-hexosaminidase, arylsulfatase, and alpha-L-iduronidase. Under conditions which disrupt lysosomes the release of the latter soluble lysosomal enzymes was demonstrated from MBR 39 bound organelles. Immunoblots of MBR 39 with purified fibroblast lysosomal membrane, demonstrated reactivity with polypeptides of molecular mass 63 kDa (major species) and 73 kDa (minor species).

Antibodies, Monoclonal↗
PubMed1997

Lysosomal biogenesis in lysosomal storage disorders.

Lysosomal biogenesis is an orchestration of the structural and functional elements of the lysosome to form an integrated organelle and involves the synthesis, targeting, functional residence, and turnover of the proteins that comprise the lysosome. We have investigated lysosomal biogenesis during the formation and dissipation of storage vacuoles in two model systems. One involves the formation of sucrosomes in normal skin fibroblasts and the other utilizes storage disorder-affected skin fibroblasts; both of these systems result in an increase in the size and the number of lysosomal vacuoles. Lysosomal proteins, beta-hexosaminidase, alpha-mannosidase, N-acetylgalactosamine-4-sulfatase, acid phosphatase, and the lysosome-associated membrane protein, LAMP-1, were shown to be elevated between 2- and 28-fold above normal during lysosomal storage. Levels of mRNA for the lysosome-associated membrane proteins LAMP-1 and LAMP-2, N-acetylgalactosamine-4-sulfatase, and the 46- and 300-kDa mannose-6-phosphate receptors were also elevated 2- to 8-fold. The up-regulation of protein and mRNA lagged 2-4 days behind the formation of lysosomal storage vacuoles. Correction of storage, in both systems, resulted in the rapid decline of the mRNA to basal levels, with a slower decrease in the levels of lysosomal proteins. Lysosomal biogenesis in storage disorders is shown to be a regulated process which is partially controlled at, or prior to, the level of mRNA. Although lysosomal proteins were differentially regulated, the coordination of these events in lysosomal biogenesis would suggest that a common mechanism(s) may be in operation.

Antibodies, Monoclonal↗
PubMed1997

Protein processing: a role in the pathophysiology of genetic disease.

Genetic diseases associated with an enzyme deficiency frequently have reduced intracellular levels of the mutant protein, despite apparently normal levels of message and protein synthesis. It has been suggested that the endoplasmic reticulum (ER) can recognise mutant protein as incorrectly folded and invoke 'quality control' processes which cause the retention and degradation of this protein. This process may occur, even for mutations which do not abrogate protein activity, contributing directly to pathophysiology. Genetic diseases associated with defects in ER and Golgi processing proteins have also been reported and generally result in impaired processing of multiple protein products. In this review the role of the ER and Golgi in the pathogenesis of genetic diseases relating to the vacuolar network are discussed.

Enzymes↗
PubMed1997

Preparation of excipient-free recombinant human tissue-type plasminogen activator by lyophilization from ammonium bicarbonate solution: an investigation of the two-stage sublimation phenomenon.

Dry, excipient-free recombinant human tissue-type plasminogen activator (tPA) powder was prepared by lyophilization from ammonium bicarbonate solution. Ammonium bicarbonate sublimes into ammonia, water, and carbon dioxide upon lyophilization, without causing measurable harm to the protein. There were approximately 4 mol of residual ammonium ion per mole of lyophilized tPA. Under certain lyophilization conditions, a large pressure increase in the lyophilizer chamber occurred, presenting a pressure control problem. Microscopy and sublimation rate measurements on the frozen matrix revealed that ice sublimation occurred first, followed by the sublimation of ammonium bicarbonate. Analysis of the sectioned frozen matrix indicated that the bicarbonate salt was evenly distributed throughout the vial, suggesting that the delay of ammonium bicarbonate sublimation was not due to hindrance by ice. In the two-stage process, ice sublimation proceeded according to zero-order kinetics, whereas ammonium bicarbonate sublimation followed a grain-burning (2/ 3-order) model and was governed by a higher activation enthalpy. In most cases, the sublimation rate of ammonium bicarbonate in the presence of tPA was lower than that in the absence of the protein. Sublimation activation enthalpy for ammonium bicarbonate in the presence of tPA was 26.1 +/- 3.8 kcal/mol, which was approximately 10 kcal/mol greater than that for the tPA-free system. Consistent with a prediction from our kinetic modeling, a 6-h extension of primary drying enabled us to conduct lyophilization while maintaining pressure control.

Bicarbonates↗
PubMed1997

Diagnosis of lysosomal storage disorders: evaluation of lysosome-associated membrane protein LAMP-1 as a diagnostic marker.

Early diagnosis of lysosomal storage disorders (LSDs), before the onset of irreversible pathologies, will be a key factor in the development of effective therapies for many of these disorders. Newborn screening offers a potential mechanism for the early detection of these disorders. From studies of both normal and LSD-affected human skin fibroblasts we identified the lysosome-associated membrane protein LAMP-1 as a potential diagnostic marker. We have developed a sensitive method for the quantification of this protein with a time-resolved fluorescence immunoassay. A soluble form of LAMP-1 was observed in plasma samples, and determination of 152 unaffected individuals gave a median value of 303 micrograms/L with the 5th and 95th percentile at 175 and 448 micrograms/L respectively. Plasma samples from 320 LSD-affected individuals representing 25 different disorders were assayed. We observed that 17 of the 25 disorder groups tested had > 88% of individuals above the 95th percentile of the control population, with 12 groups having 100% above the 95th percentile. Overall, 72% of patients had LAMP-1 concentrations above the 95th percentile of the unpartitioned control population. We suggest that LAMP-1 may be a useful marker in newborn screening for LSDs.

Adolescent↗
PubMed1996

Enzyme replacement therapy in a feline model of Maroteaux-Lamy syndrome.

We report studies that suggest enzyme replacement therapy will result in a significant reduction in disease progression and tissue pathology in patients with Maroteaux-Lamy syndrome (Mucopolysaccharidosis type VI, MPS VI). A feline model for MPS VI was used to evaluate tissue distribution and clinical efficacy of three forms of recombinant human N-acetylgalactosamine-4-sulfatase (rh4S, EC 3.1.6.1). Intravenously administered rh4S was rapidly cleared from circulation. The majority of rh4S was distributed to liver, but was also detected in most other tissues. Tissue half-life was approximately 2-4 d. Three MPS VI cats given regular intravenous infusions of rh4S for up to 20 mo showed variable reduction of storage vacuoles in Kupffer cells and connective tissues, however cartilage chondrocytes remained vacuolated. Vertebral bone mineral volume was improved in two MPS VI cats in which therapy was initiated before skeletal maturity, and increased bone volume appeared to correlate with earlier age of onset of therapy. One cat showed greater mobility in response to therapy.

Animals↗
PubMed1996

Identification, expression, and biochemical characterization of N-acetylgalactosamine-4-sulfatase mutations and relationship with clinical phenotype in MPS-VI patients.

Maroteaux-Lamy syndrome, or mucopolysaccharidosis type VI (MPS-VI), is a lysosomal storage disorder characterized by the defective degradation of dermatan sulfate due to the deficiency of N-acetylgalactosamine-4-sulfatase (4S). The clinical severity of MPS-VI ranges in a continuum from mildly affected to severely affected patients. Mutations in MPS-VI patient samples were identified by chemical cleavage and direct DNA sequencing of PCR products derived from patient cDNA. Five amino acid substitutions were identified (T92M, R95Q, Y210C, H393P, and L498P), individually introduced into the wild-type 4S cDNA by site-directed in vitro mutagenesis, and transfected into Chinese hamster ovary cells. Three of the five mutations (R95Q, Y210C, and H393P) were observed in >1 of 25 unrelated MPS-VI patients; however, the mutations were not found in 20 control individuals. The residual 4S activity and protein (biochemical phenotype) were determined for each mutant in order to confirm their identity as mutations and to dissect the contribution of each mutant allele to the overall clinical phenotype of the patient. For each patient, the combined biochemical phenotypes of the two 4S mutant alleles demonstrated a good correspondence with the observed clinical phenotype (with the possible exception of a patient who was a compound heterozygote for T92M and L498P). This preliminary correspondence between genotype and the phenotype in MPS-VI may, with further refinement, contribute to the assessment of therapeutic approaches for MPS-VI patients.

Adolescent↗
PubMed1995

Two site-directed mutations abrogate enzyme activity but have different effects on the conformation and cellular content of the N-acetylgalactosamine 4-sulphatase protein.

The sulphatase family of enzymes have regions of sequence similarity, but relatively little is known about either the structure-function relationships of sulphatases, or the role of highly conserved amino acids. The sequence of amino acids CTPSR at position 91-95 of 4-sulphatase has been shown to be highly conserved in all of the sequenced sulphatase enzymes. The cysteine at amino acid 91 of 4-sulphatase was selected for mutation analysis due to its potential role in either the active site, substrate-binding site or part of a key structural domain of 4-sulphatase and due to the absence of naturally occurring mutations in this residue in mucopolysaccharidosis type VI (MPS VI) patients. Two mutations, C91S and C91T, altering amino acid 91 of 4-sulphatase were generated and expressed in Chinese hamster ovary cells. Biochemical analysis of protein from a C91S cell line demonstrated no detectable 4-sulphatase enzyme activity but a relatively normal level of 4-sulphatase polypeptide (180% of the wild-type control protein level). Epitope detection, using a panel of ten monoclonal antibodies, demonstrated that the C91S polypeptide had a similar immunoreactivity to wild-type 4-sulphatase, suggesting that the C91S substitution does not induce a major structural change in the protein. Reduced catalytic activity associated with normal levels of 4-sulphatase protein have not been observed in any of the MPS VI patients tested and all show evidence of structural modification of 4-sulphatase protein with the same panel of antibodies [Brooks, McCourt, Gibson, Ashton, Shutter and Hopwood (1991) Am. J. Hum. Genet. 48, 710-719]. The loss of enzyme activity without a detectable protein conformation change suggests that Cys-91 may be a critical residue in the catalytic process. In contrast, analysis of protein from a C91T cell line revealed low levels of catalytically inactive 4-sulphatase polypeptide (0.37% of the wild-type control protein level) which had missing or masked epitopes, suggesting an altered protein structure or conformation. Subcellular fractionation studies of the C91T cell line demonstrated a high proportion of 4-sulphatase polypeptide content in organelles characteristic of microsomes. The aberrant intracellular localization and the reduced cellular content of 4-sulphatase polypeptide was consistent with the observed structural modification leading to retention and degradation of the protein within an early vacuolar compartment.

Animals↗
PubMed1995

Surface denaturation at solid-void interface--a possible pathway by which opalescent particulates form during the storage of lyophilized tissue-type plasminogen activator at high temperatures.

During protein lyophilization, it is common practice to complete the freezing step as fast as possible in order to avoid protein denaturation, as well as to obtain a final product of uniform quality. We report a contradictory observation made during lyophilization of recombinant tissue-type plasminogen activator (t-PA) formulated in arginine. Fast cooling during lyophilization resulted in a lyophilized product that yielded more opalescent particulates upon long term storage at 50 degrees C, under a 150 mTorr nitrogen seal gas environment. Fast cooling also resulted in a lyophilized cake with a large internal surface area. Studies on lyophilized products containing 1% (w/w) residual moisture and varying cake surface areas (0.22-1.78 m2/gm) revealed that all lyophilized cakes were in an amorphous state with similar glass transition temperatures (103-105 degrees C). However, during storage the rate of opalescent particulate formation in the lyophilized product (as determined by UV optical density measurement in the 360 to 340 nm range for the reconstituted solution) was proportional to the cake surface area. We suggest that this is a surface-related phenomenon in which the protein at the solid-void interface of the lyophilized cake denatures during storage at elevated temperatures. Irreversible denaturation at the ice-liquid interface during freezing in lyophilization is unlikely to occur, since repeated freezing/thawing did not show any adverse effect on the protein. Infrared spectroscopic analysis could not determine whether protein, upon lyophilization, at the solid-void interface would still be in a native form.

Calorimetry, Differential Scanning↗