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Biomedical subjects

G A Grabowski

Publications and source records attributed to G A Grabowski.

At least 55 records · Page 3Linked to original sources
PubMed1996

Proteolytic processing patterns of prosaposin in insect and mammalian cells.

Prosaposin is a multifunctional protein encoded at a single locus in humans and mice. The precursor contains, in tandem, four glycoprotein activators or saposins, termed A, B, C, and D, that are essential for specific glycosphingolipid hydrolase activities. Prosaposin appears to be a potent neurotrophic factor. To explore the proteolytic processing from prosaposin to mature activator proteins, metabolic labeling was done with human prosaposin expressed in insect cells, human fibroblasts, neuronal stem cells (NT2) and retinoic acid-differentiated NT2 neurons. In all cell types, the major processing pathway was through a tetrasaposin, A-B-C-D, from which saposin A was then removed. In mammalian cells monosaposins were derived from the trisaposin B-C-D by cleavage to the disaposins, B-C and C-D, that were processed to monosaposins. In insect cells the major end products were the disaposins, with A-B and C-D derived from the tetrasaposin, A-B-C-D, or with B-C and C-D derived from the trisaposin, B-C-D. In insect and mammalian cells, the nonsignal NH2-terminal peptide preceding saposin A (termed Nter) was usually removed prior to saposin A cleavage. In NT2-derived differentiated neurons, precursor tetrasaposins containing A-B-C-D were secreted with and without Nter. Immunofluorescence studies using prosaposin-specific antisera showed large steady state amounts of uncleaved prosaposin in Purkinje cells, cortical neurons, and other specific cell types in adult mice. These studies indicate that prosaposin processing is highly regulated at a proteolytic level to produce prosaposin, tetrasaposins, or mature monosaposins in specific mammalian cells.

Animals↗
PubMed1996

Bioproduction of human enzymes in transgenic tobacco.

Transgenic plants have significant potential in the bioproduction of complex human therapeutic proteins due to ease of genetic manipulation, lack of potential contamination with human pathogens, conservation of eukaryotic cell machinery mediating protein modification, and low cost of biomass production. Tobacco has been used as our initial transgenic system because Agrobacterium-mediated transformation is highly efficient, prolific seed production greatly facilitates biomass scale-up, and development of new "health-positive" uses for tobacco has significant regional support. We have targeted bioproduction of complex recombinant human proteins with commercial potential as human pharmaceuticals. Human protein C (hPC), a highly processed serum protease of the coagulation/anticoagulation cascade, was produced at low levels in transgenic tobacco leaves. Analogous to its processing in mammalian systems, tobacco-synthesized hPC appears to undergo multiple proteolytic cleavages, disulfide bond formation, and N-linked glycosylation. Although tobacco-derived hPC has not yet been tested for all posttranslational modifications or for enzymatic (anticlotting) activity, these results are promising and suggest considerable conservation of protein processing machinery between plants and animals. CropTech researchers have also produced the human lysosomal enzyme glucocerebrosidase (hGC) in transgenic tobacco. This glycoprotein has significant commercial potential as replacement therapy in patients with Gaucher's disease. Regular intravenous administration of modified glucocerebrosidase, derived from human placentae or CHO cells, has proven highly effective in reducing disease manifestations in patients with Gaucher's disease. However, the enzyme is expensive (dubbed the "world's most expensive drug" by the media), making it a dramatic model for evaluating the potential of plants to provide a safe, low-cost source of bioactive human enzymes. Transgenic tobacco plants were generated that contained the human glucocerebrosidase cDNA under the control of an inducible plant promoter. hGC expression was demonstrated in plant extracts by enzyme activity assay and immunologic cross-reactivity with anti-hGC antibodies. Tobacco-synthesized hGC comigrates with human placental-derived hGC during electrophoretic separations, is glycosylated, and, most significantly, is enzymatically active. Although expression levels vary depending on transformant and induction protocol, hGC production of > 1 mg/g fresh weight of leaf tissue has been attained in crude extracts. Our studies provide strong support for the utilization of tobacco for high-level production of active hGC for purification and eventual therapeutic use at potentially much reduced costs. Furthermore, this technology should be directly adaptable to the production of a variety of other complex human proteins of biologic and pharmaceutical interest.

Enzymes↗
PubMed1996

Functional organization of saposin C. Definition of the neurotrophic and acid beta-glucosidase activation regions.

Saposin C is an essential co-factor for the hydrolysis of glucosylceramide by acid beta-glucosidase in mammals. In addition, prosaposin promotes neurite outgrowth in vitro via sequences in saposin C. The regional organization of these neurotrophic and activation properties of saposin C was elucidated using recombinant or chemically synthesized saposin Cs from various regions of the molecule. Unreduced and reduced proteins were analyzed by electrospray-mass spectrometry to establish the complement of disulfide bonds in selected saposin Cs. Using saposin B as a unreactive backbone, chimeric saposins containing various length segments of saposin B and C localized the neurotrophic and acid beta-glucosidase activation properties to the carboxyl- and NH2-terminal 50% of saposin C, respectively. The peptide spanning residues 22-31 had neurotrophic effects. Molecular modeling and site-directed mutagenesis localized the activation properties of saposin C to the region spanning residues 47-62. Secondary structure was needed for retention of this property. Single substitutions of R and S at the conserved cysteines at 47 or 78 diminished but did not obliterate the activation properties. These results indicate the segregation of neurotrophic and activation properties of saposin C to two different faces of the molecule and suggest a topographic sequestration of the activation region of prosaposin for protection of the cell from adverse hydrolytic activity of acid beta-glucosidase.

Amino Acid Sequence↗
PubMed1996

Gaucher disease: identification of three new mutations in the Korean and Chinese (Taiwanese) populations.

Gaucher Disease type 1, the most prevalent lysosomal disease among Caucasians, is due to defects in the activity of acid beta-glucosidase. Over 40 missense, nonsense, and more complex alleles have been described, primarily in Western populations. From these results, predictive genotype/phenotype correlations have been developed and used to guide genetic counseling and therapy. Only a few mutations have been described in Japanese patients with Gaucher disease and many of these have resulted in severe phenotypes. Although rare, Gaucher Disease occurs in Korean and Chinese (Taiwanese) populations. Sequencing of RT-PCR cDNAs from five unrelated Korean and two sibling Chinese (Taiwanese) Gaucher type 1 patients identified three new Gaucher disease mutations. These disease alleles encoded V15L, G46E, and N188S substitutions leading to dysfunctional acid beta-glucosidases. The G46E was present in two Korean patients and the N188S allele was present in the Korean and Chinese (Taiwanese) populations, suggesting an ancient mutation. The commonality of these two mutations in the Korean and Chinese (Taiwanese) population indicates the need for more extensive screening for these mutations in the Gaucher populations.

Adolescent↗
PubMed1996

Turnover and distribution of intravenously administered mannose-terminated human acid beta-glucosidase in murine and human tissues.

Gaucher disease type 1, the most prevalent lysosomal storage disease, is caused by the defective activity of the lysosomal enzyme, acid beta-glucosidase, or glucocerebrosidase. Infusion of purified acid beta-glucosidase containing alpha-mannosyl-terminated oligosaccharides (alglucerase) is efficacious in reversing hematologic, hepatic, splenic, and bony disease manifestations. The murine tissue distribution and turnover of bolus injections of alglucerase was evaluated by enzymatic activity, quantitative cross-reacting immunologic material analyses, and immunofluorescence studies. Enzyme activity measurements detected distribution to liver, spleen, thymus, kidney, and bone marrow mononuclear cells, but not to lungs and brain. In kidney and thymus, the enzyme was transiently present. In liver and spleen, enzyme activity peaked at about 20 min postinjection followed by a biphasic decrease with t1/2 approximately 40-60 min and approximately 12-14 h. In bone marrow maximal enzyme activity was at 40-60 min with a disappearance t1/2 approximately 60 min. Quantitative cross-reacting immunologic material studies of liver and spleen showed delivery of enzyme with decreased catalytic rate constants whose degradation included denaturation and proteolytic components. By immunofluorescence the human enzyme was distributed primarily to reticuloendothelial cells of the liver and spleen. In autopsy material from a Gaucher disease type 2 patient treated with enzyme, immunohistochemical and activity studies showed distributions similar to those in mice. These studies indicate a complex delivery and intracellular degradation of acid beta-glucosidase with lower intrinsic activity than the administered therapeutic agent.

Animals↗
PubMed1996

Current issues in enzyme therapy for Gaucher disease.

Enzyme therapy has altered forever the management of patients with Gaucher disease. Studies in over 1200 treated Gaucher disease patients have demonstrated regression of hepatic, splenic, bony and haematological abnormalities, with a return towards health in many affected patients. The therapy is well tolerated, with approximately 7% of patients having adverse effects. However, the lack of standardised clinical staging and tracking procedures, and a poor understanding of the basic biochemistry and cell biology of the administered enzyme, continue to inhibit optimisation of treatment. Ultimately, preventive intervention with enzyme therapy will require absolute safety and much less expensive preparations, and accurate predictive genotype testing to fully optimise this mode of therapy. The success and pitfalls encountered in enzyme therapy for Gaucher disease provide a map for the development of such therapies for other inborn errors of metabolism.

Gaucher Disease↗
PubMed1996

Tissue and cellular specific expression of murine lysosomal acid lipase mRNA and protein.

Lysosomal acid lipase (LAL) is essential to the intracellular control of cholesterol and triglyceride catabolism via the low density lipoprotein (LDL) delivery of these neutral lipids to the lysosome. Deficiency of LAL in humans leads to Wolman disease and cholesteryl ester storage disease that result, respectively, in the intralysosomal storage of both neutral lipids or only cholesteryl esters. The mouse and human LAL cDNAs were cloned. The deduced amino acid sequences from the mouse and human LAL had high similarity (95%) and identity (75%) including conservation of the active center motifs (G-X-S-X-G) and five potential N-glycosylation consensus sequences. Tissue specific expression of LAL mRNA and protein in mouse tissues was evaluated by in situ hybridization and immunofluorescence staining, respectively. The LAL mRNA was expressed at low levels in most tissues. High level expression was found in hepatocytes and splenic and thymic cells. Very high level expression was observed in cells of the small intestinal villi, the zona fasciculata and reticularis of the adrenal cortex, pancreatic acini, and renal tubular epithelium. Significant levels of expression were detected in epithelial cells of choroid plexus in developing mouse embryo by day 12, in liver and lung by day 14, and in small intestine and kidney by day 16. Similar distribution of LAL protein was observed by immunofluorescence stain. Our results show that the expression of LAL is regulated in a tissue- and cell-specific manner that corresponds to the pathologic involvement in Wolman disease.-Du, H., D. P. Witte, and G. A. Grabowski. Tissue and cellular specific expression of murine lysosomal acid lipase mRNA and protein.

Amino Acid Sequence↗
PubMed1995

Characterization of lysosomal acid lipase by site-directed mutagenesis and heterologous expression.

Lysosomal acid lipase (LAL) is essential for the hydrolysis of cholesterol esters and triglycerides that are delivered to the lysosomes via the low density lipoprotein receptor system. The deficiency of LAL is associated with cholesteryl ester storage disease (CESD) and Wolman's disease (WD). We cloned the human LAL cDNA and expressed the active enzyme in the baculovirus system. Two molecular forms (M(r) approximately 41,000 and approximately 46,000) with different glycosylation were found intracellularly, and approximately 24% of the M(r) approximately 46,000 form was secreted into the medium. Tunicamycin treatment produced only an inactive M(r) approximately 41,000 form. This result implicates glycosylation occupancy in the proper folding for active-site function. Catalytic activity was greater toward cis- than trans-unsaturated fatty acid esters of 4-methylumbelliferone and toward esters with 7-carbon length acyl chains. LAL cleaved cholesterol esters and mono-, tri-, and diglycerides. Heparin had a biphasic effect on enzymatic activity with initial activation followed by inhibition. Inhibition of LAL activity by tetrahydrolipstatin and diethyl p-nitrophenyl phosphate suggested the presence of active serines in binding/catalytic domain(s) of the protein. Site-directed mutagenesis at two putative active centers, GXSXG, showed that Ser153 was important to catalytic activity, whereas Ser99 was not and neither was the catalytic nucleophile. Three reported mutations (L179P, L336P, and delta AG302 deletion) from CESD patients were created and expressed in the Sf9 cell system. None cleaved cholesterol esters, and L179P and L336P cleaved only triolein at approximately 4% of wild-type levels. These results suggest that mechanisms, in addition to LAL defects, may operate in the selective accumulation of cholesterol esters or triglycerides in CESD and WD patients.

Animals↗
PubMed1995

Enzyme therapy in type 1 Gaucher disease: comparative efficacy of mannose-terminated glucocerebrosidase from natural and recombinant sources.

OBJECTIVE: To compare the efficacy of mannose-terminated glucocerbrosidase prepared from natural (alglucerase; Ceredase, Genzyme Corp., Cambridge, Massachusetts) and recombinant (imiglucerase; Cerezyme, Genzyme Corp.) sources in treating type 1 Gaucher disease. DESIGN: Double-blind, randomized, parallel trial. SETTING: University medical center and clinical research hospital. PATIENTS: 15 patients (4 children and 11 adults) randomly assigned to receive Ceredase and 15 patients (3 children and 12 adults) assigned to receive Cerezyme. INTERVENTION: Ceredase and Cerezyme were infused every 2 weeks for 9 months at a dose of 60 U/kg body weight. OUTCOME MEASURES: Hemoglobin levels, platelet counts, and serum acid phosphatase and angiotensin-converting enzyme activities were monitored every 2 weeks during the trial. Hepatic and splenic volumes were assessed at the time of randomization and after 6 and 9 months of enzyme infusion. Formation of IgG antibodies to Ceredase or Cerezyme was monitored every 3 months by radioimmunoprecipitation assay. RESULTS: No significant differences were found in the rate or extent of improvement in hemoglobin levels, platelet counts, serum acid phosphatase or angiotensin-converting enzyme activities, or hepatic or splenic volumes between either treatment group. The incidence of IgG antibody formation was greater in the Ceredase group (40%) than in the Cerezyme group (20%). No major immunologic adverse events occurred in either group. CONCLUSIONS: Our study shows the therapeutic similarity of Ceredase and Cerezyme. Cerezyme has the advantage of being theoretically unlimited in supply and free of potential pathogenic contaminants.

Adolescent↗
PubMed1995

Pathological findings in Gaucher disease type 2 patients following enzyme therapy.

The pathological outcomes following intravenous acid beta-glucosidase (alglucerase) infusions were compared in two siblings with Gaucher disease type 2, the acute neuronopathic variant. In case 1 enzyme infusions (four doses at 7 months) had no effect when severe progressive visceral and neuronopathic disease were present. Death from progressive disease occurred at 9 months. Case 2 was prenatally diagnosed. Enzyme infusions were initiated presymptomatically at 4 days of age and continued until death at 15.2 months. Development progressed satisfactorily, albeit at a slower than normal rate until age 10 months when progressive brain stem involvement became evident. Death occurred after slowly progressive brain stem dysfunction, but gross motor and cognitive skills were nearly normal. Postmortem light and electron microscope (EM) studies in both cases showed typical central nervous system (CNS) findings and massive infiltration of the lungs and lymph nodes by Gaucher cells. The liver, spleen, and bone marrow, except that in the temporal bone, in case 2 were normal. These studies show that enzyme therapy may slow but does not prevent the development of lethal CNS disease in Gaucher disease type 2, even when initiated presymptomatically. These findings also indicate the nonuniformity of tissue responses to enzyme therapy implying the existence of therapeutically inaccessible compartments that, in less severe variants, may create unexpected long-term disease complications.

Fatal Outcome↗
PubMed1995

Effect of cellular type on expression of acid beta-glucosidase: implications for gene therapy in Gaucher disease.

The effects of cellular type on the expressed activity of acid beta-glucosidase were evaluated using retroviral constructs containing the human cDNA. MFG retroviral ecotropic and amphotropic vectors containing the human acid beta-glucosidase cDNA were produced and used to infect different murine cell lines (fibroblast, neuronal and monocytic) and human cells (HL60 and cord blood CD34+), respectively. The expression of human acid beta-glucosidase was evaluated by enzyme activity assays, quantitative Western blots and immunofluorescence. All cells permanently integrated viruses and expression of enzyme protein was achieved in all cell lines, but cellular transduction efficiency differed even between different neuronal cell lines (eg N18S > PC12). In most cell lines acid beta-glucosidase activity was increased between two- and three-fold with concomitant signal increases by Western blot and immunofluorescence N18S cells had poor transduction efficiency, but high cellular expression in transduced cells. In NIH3T3 and MC3T3-E1 cells acid beta-glucosidase protein was expressed in 2-, 7- and 14-day cultures after infection and at least to passage four. The expressed acid beta-glucosidase in NIH3T3 cells was at two to three times normal activity levels, and was processed similarly to the human fibroblast enzyme. Inactive human acid beta-glucosidase was expressed in MC3T3-E1 preosteoblastic cells and this was maintained during differentiation to osteoblasts. These results indicate that gene transfer results in cell lines may not be generally extrapolated to all cells in tissues or to differentiated progeny.

3T3 Cells↗
PubMed1994

Functional human saposins expressed in Escherichia coli. Evidence for binding and activation properties of saposins C with acid beta-glucosidase.

Small (80-amino acid) glycoproteins or saposins are important for the in vivo function of several lysosomal hydrolases. Four saposins, A, B, C, and D, are encoded by a single locus termed prosaposin. Saposins C and A are thought to function in vivo as activators of acid beta-glucosidase. The physiologic role of saposin C has been confirmed, whereas that of saposin A role has not. To investigate the effects of saposins C and A on acid beta-glucosidase activity, the coding sequence for the individual saposins was expressed in Escherichia coli and the recombinant proteins purified to homogeneity. Recombinant and natural saposins A and C activated acid beta-glucosidase similarly only in micromolar amounts. Saposin C had specific activation of acid beta-glucosidase activity at < 200 nM. A second phase of activation was achieved at > 1 microM. In comparison, saposin A consistently activated acid beta-glucosidase only at > 1 microM. Two mutant saposins C (Cys382-->Phe and Cys382--Gly) were created and shown to compete with saposin C for a site on acid beta-glucosidase. The mutant saposins did not activate the enzyme. Recombinant saposin A (< 200 nM) competed with saposin C for a site on the enzyme but without activating effects. These studies show that saposin A is not an in vitro activator of acid beta-glucosidase at physiologic concentrations, although binding occurs without activating acid beta-glucosidase. The studies with mutant saposins C indicate that the binding and activation effects of saposins C are distinct events. These results indicate that the saposin C-induced conformational change in the enzyme occurs via highly specific, probably multivalent, interactions between acid beta-glucosidase and saposin C.

Amino Acid Sequence↗
PubMed1994

Identification of Glu340 as the active-site nucleophile in human glucocerebrosidase by use of electrospray tandem mass spectrometry.

Gaucher disease is an inherited lysosomal storage disorder caused by a deficiency of human acid beta-glucosidase (glucocerebrosidase). This enzyme is inactivated by the specific, mechanism-based enzyme inactivator 2-deoxy-2-fluoro-beta-D-glucopyranosyl fluoride, which functions by forming a stable 2-deoxy-2-fluoro-alpha-D-gluco-pyranosyl-enzyme intermediate. The key nucleophilic amino acid residue involved in formation of this intermediate was conclusively identified by tandem mass spectrometry as Glu340, and not Asp443 as thought previously. This was confirmed by site-directed mutagenesis. Identification, and mass determination, of the labeled peptide in a proteolytic hydrolysate involved detection of a collision-induced fragmentation reaction specific to the sugar-peptide linkage. Confirmation of the identity of the labeled peptide was obtained both by tandem mass spectrometric sequencing and by chemical degradation of the purified peptide. This method allowed the rapid, sensitive, and non-isotopic determination of an essential amino acid residue in a clinically important enzyme.

Amino Acid Sequence↗
PubMed1994

Analysis of human acid beta-glucosidase by site-directed mutagenesis and heterologous expression.

Structure/function relationships of acid beta-glucosidase, the enzyme deficient in Gaucher disease, were evaluated by characterizing the proteins expressed from cDNAs encoding normal and mutant enzymes. Twenty-two Gaucher disease mutations or created mutations were expressed in Spodoptera frugiperda (Sf9) cells and analyzed for catalytic properties, stability, inhibitor binding, and modifier interactions. Many Gaucher disease mutations encoded highly disruptive amino acid substitutions (e.g. P289L and D409V) and produced severely compromised proteins with very reduced activity (kcat < 1% of normal) and/or stability. Six mutant enzymes had sufficient catalytic activity (kcat approximately 5-30% of normal) for extensive studies. The highly conservative substitutions, i.e. F216Y or S364T and V394L, led to severe, but selective, abnormalities of enzyme stability or large decreases in catalytic activity, respectively. The T323I, N370S, and V394L enzymes interacted abnormally with active site-directed inhibitors and localized these residues to the glycon binding region. Selected mutant enzymes were poorly activated by phosphatidylserine (V394L, L444P, and R463C) or by saposin C (L444P and T323I), indicating that the enzyme sites for interaction with these activators were within the carboxyl one-third of the enzyme. Substitutions of Ser, Glu, and/or Gly at residues Asp-443 and/or Asp-445 demonstrated important steric roles for these residues in the active site, but neither is the catalytic nucleophile. Together with previous studies, the present analyses provide an insight into the pathogenesis of Gaucher disease and the functional organization of acid beta-glucosidase.

Amino Acid Sequence↗
PubMed1994

Developmental and tissue-specific expression of prosaposin mRNA in murine tissues.

Prosaposin is a multifunctional locus in humans and mice that encodes in tandem and in the same reading frame four glycoprotein activators, or saposins, of lysosomal hydrolases. These ubiquitously expressed glycoproteins and the precursor, prosaposin, have been proposed to function in glycosphingolipid catabolic pathways and glycolipid transport. To characterize the temporal and spatial expression of the prosaposin locus, prenatal and postnatal mouse tissues were screened by in situ hybridization with a mouse antisense riboprobe for prosaposin. Prenatally, prosaposin mRNA was expressed differentially in the placenta and prominently in the decidua basalis and capsularis where expression was gestational age dependent. No other region of high-level expression was detectable in the prenatal mouse. In comparison, high-level differential expression of prosaposin was clearly evident postnatally in a variety of organs, including secretory epithelial cells of the choroid plexus, ependymal lining, upper trachea, esophagus, cortical tubules of the kidney, sertoli cells of the testes and epididymis. Discrete localization of prosaposin mRNA expression was also found in neurons of the cerebral cortex, cerebellar cortex, and lateral columns of the spinal cord as well as in hepatocytes of the mature liver. Very high levels of expression were found in specialized tissues including the Harderian glands and macrophages of lymph nodes, lungs, splenic tissue, and thymus. These studies indicate that the expression of the prosaposin locus, a presumed "housekeeping" gene, is under tissue- and cell-specific differential control. The spatial organization of expression suggests a role for this locus in the expression of glycosphingolipid-storage diseases.

Animals↗
PubMed1993

Synthesis and use of novel fluorescent glycosphingolipids for estimating beta-glucosidase activity in vitro in the absence of detergents and subtyping Gaucher disease variants following administration into intact cells.

Two novel fluorescent glycolipids, LRO-glucosylceramide (LRO-GC) and LRO-trihexosylceramide (LRO-THC) were synthesized and utilized for estimating activities of the lysosomal, acid beta-glucosidase in cell extracts and intact skin fibroblasts, derived from normal individuals and patients with Gaucher disease subtypes. The uniqueness of the glycolipids is the fact that a fluorescent probe (lissamine rhodamine) is linked in a sulfonylamide linkage to the sphingosyl residue of the sphingolipid. Thus, the product of enzymatic hydrolysis, lissamine rhodamine sulfonylamido sphingosine (LRO-ceramide) cannot be further hydrolyzed and remains a metabolic end product. A unique property of LRO-GC as a substrate for the lysosomal, acid beta-glucosidase in vitro was the observation that enzymatic hydrolysis occurs in the absence of detergents and that hydrolytic rates are, in fact, reduced in the presence of Triton X-100 and/or sodium taurocholate. Also, both glycolipids penetrated the membrane of intact fibroblasts in the absence of serum and were hydrolyzed in lysosomes of the intact cells. The rates of intracellular hydrolysis decreased with the severity of the Gaucher disease subtypes. Using LRO-THC as substrate, the intracellular ratio of LRO-ceramide to LRO-glucosylceramide was an indicator for the specific GD-subtype.

Cells, Cultured↗