| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Articles |
-Glucosidase Protein: Evaluation as a Screening Marker for Pompe Disease and Other Lysosomal Storage Disorders
1
Lysosomal Diseases Research Unit and
2
State Screening Services, Department of Chemical Pathology, Women’s and Children’s Hospital, 72 King William Rd., North Adelaide, South Australia 5006, Australia.
3
Department of Clinical Genetics, Erasmus University, PO
Box 1738, 3000 DR Rotterdam, The Netherlands.
a Author for correspondence. Fax 61-8-8204-7100; e-mail pmeikle{at}medicine.adelaide.edu.au
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-glucosidase
protein was evaluated as a marker protein for Pompe disease and
potentially for other LSDs.
Methods: Two sensitive immunoquantification assays for the
measurement of total (precursor and mature) and mature forms of acid
-glucosidase protein were used to determine the concentrations in
plasma and dried blood spots from control and LSD-affected individuals.
Results: In the majority of LSDs, no significant increases above
control values were observed. However, individuals with Pompe disease
showed a marked decrease in acid -glucosidase protein in both plasma
and whole blood compared with unaffected controls. For plasma samples,
this assay gave a sensitivity of 95% with a specificity of 100%. For
blood spot samples, the sensitivity was 82% with a specificity of
100%.
Conclusions: This study demonstrates that it is possible to
screen for Pompe disease by screening the concentration of total acid
-glucosidase in plasma or dried blood spots.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-glucosidase. Patients with Pompe
disease are unable to degrade glycogen stored in the lysosome, leading
to the accumulation of this substrate in lysosomal storage vacuoles.
Morphologically, this produces an increase in the size and number of
lysosomes in the cell. Pompe disease can present as infantile,
juvenile, or adult onset forms. The infantile onset form is
characterized by massive cardiomegaly, macroglossia, progressive muscle
weakness (including respiratory muscles), and marked hypotonia, with
death occurring within the first 2 years of life. The juvenile and
adult onset forms manifest as slower progressive muscular disorders
that are limited to skeletal muscle, with death usually occurring from
respiratory failure (1). The heterogeneous presentation of
Pompe disease results, at least in part, from the occurrence of
different mutations in the lysosomal acid -glucosidase gene, which
can lead to variable effects on the functional capacity of the mutant
enzyme.
According to Wisselaar et al. (2), lysosomal acid
-glucosidase (EC 3.2.1.3) is synthesized as a precursor protein with
molecular mass of 110 kDa, which is then transported from the
endoplasmic reticulum to the trans Golgi network. A large proportion of
precursor molecule is transported to the lysosomes where it is
proteolytically converted to 76- and 70-kDa forms via a long-lived
intermediate molecule of 95 kDa. A small amount of precursor protein is
transported to the plasma membrane and secreted.
Pompe disease is one of >40 distinct genetic diseases known
collectively as lysosomal storage disorders (LSDs). LSDs have a
combined incidence of 
1 in 5000 births (3). On the basis
of clinical diagnosis, Pompe disease has a reported incidence of 1 in
201 000 births in the Australian population (4); however,
recent studies based on carrier detection in the general
population have indicated that the incidence is much greater, at 1
in 40 000 births, in both the US (5) and The Netherlands
(6).
Definitive treatment for Pompe disease is not currently available.
However, two main treatment strategies are being developed. Correction
of the enzyme deficiency by enzyme replacement therapy is well advanced
(7)(8), and gene therapy using viral vectors is
also under development (9)(10)(11)(12). In the quail and mouse
animal models, enzyme replacement therapy using the precursor form of
acid -glucosidase (7)(13) has led to the
clinical and metabolic correction of Pompe disease. It is anticipated
that this type of therapy will be available for human use in the near
future. Similar treatment strategies are being developed for many LSDs
(3), and it is well recognized that early diagnosis and
treatment will provide a substantial improvement in the efficacy of
these therapies for this group of disorders. However, in the absence of
a family history, the only practical way to achieve early diagnosis is
through a newborn screening program.
The clinical diagnosis of Pompe disease is confirmed by the virtual
absence (in infantile onset) or markedly reduced (in juvenile and adult
onset) activity of acid -glucosidase in muscle biopsies and cultured
fibroblasts. Prenatal diagnosis can be made by determining the acid
-glucosidase activity in cultured amniotic cells and/or in chorionic
villus biopsies (14)(15) and also by mutation
analysis (15). However, current diagnostic tools are not
suitable for large-scale screening. Immunoquantification of the acid
-glucosidase protein can be adapted for large-scale screening;
however, the protein concentration may not be diminished in all
individuals with Pompe disease. Normal amounts of enzyme, which appear
to be catalytically inactive, have been reported to be common in
infantile patients in China (16).
We previously evaluated lysosomal membrane glycoprotein-1 (LAMP-1)
(17) and LAMP-2 (18) as effective screening
markers for LSDs. Although these proteins were determined to be useful
markers for many LSDs, they showed only marginal specificity and
sensitivity for Pompe disease. In this study, we proposed to evaluate
acid -glucosidase as a marker for Pompe disease and other LSDs.
Accordingly, we developed two sensitive immunoquantification assays for
the determination of either the total (precursor and mature forms) or
mature form only of acid -glucosidase protein in dried blood spots
and plasma. We used these assays to determine the concentrations of
these proteins in plasma and dried blood spot samples taken from
unaffected and LSD-affected individuals.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Plasma samples used in this study were from samples submitted to the National Referral Laboratory for the Diagnosis of Lysosomal, Peroxisomal and Other Genetic Disorders and samples processed for routine biochemistry in the Department of Chemical Pathology, Women’s and Children’s Hospital. Additional plasma samples from Gaucher patients were obtained from Dr. Allan Cooper (Willink Biochemical Institute, Manchester, UK).
reagents
Acid -glucosidase proteins.
Pharming BV (The Netherlands)
provided recombinant precursor and mature forms of acid -glucosidase
proteins purified from rabbit (8) and mouse milk,
respectively (19). Purity of the proteins was established by
sodium dodecyl sulfate-polyacrylamide gel electrophoretic
analysis carried out on 12.5% acrylamide gels using the method of
Laemmli (20) and staining with silver (21).
Protein calibrators were quantified by the bicinchoninic acid
method of Smith et al. (22) using bovine serum
albumin as a calibrator.
Polyclonal antibodies.
Sheep anti-acid -glucosidase
polyclonal antibody was produced against the recombinant precursor form
of the protein. A sheep received subcutaneous injections containing 2
mg of protein in 1 mL of an emulsion of phosphate-buffered saline (pH
7.4) and complete Freund’s adjuvant, followed by four booster
injections (2 mg each) with incomplete Freund’s adjuvant, each 3 weeks
apart. One week after the last injection, the sheep was bled out and
serum collected.
Monoclonal antibodies.
The hybridoma cell lines producing
monoclonal antibodies that recognize both the precursor and mature
forms (43D1) (23) and the mature form only (43G8)
(24) of the acid -glucosidase protein were provided by
Pharming BV (The Netherlands).
Purification of antibodies.
Sheep polyclonal antibody was
purified on a 5-mL HitrapTM protein G affinity
column (Pharmacia Biotech) followed by an acid -glucosidase
affinity column. The acid -glucosidase affinity column was prepared
by coupling 5 mg of the precursor form of acid -glucosidase protein
to 2.5 mL of Affi-Prep Hz support (Bio-Rad) according to the
manufacturer’s instructions.
Briefly, 5 mL of sheep serum was diluted with 5 mL of
phosphate-buffered saline (pH 7.4) and centrifuged at 2200g
for 10 min at 4 °C. The centrifuged serum was passed through a 0.2
µm filter, and then loaded onto the protein G column at a flow rate
of 0.5 mL/min. The column was washed with phosphate-buffered saline (pH
7.4), and the antibody was eluted with 0.1 mol/L
H3PO4/NaH2PO4
(pH 2.5) and immediately neutralized by adding 1.0 mol/L
Na2HPO4 (1:10, by
volume). The protein content was estimated by the absorbance at
280 nm (absorbance = 1.4 for 1.0 g/L protein). The eluate was
diluted fourfold and then loaded onto the acid -glucosidase affinity
column at the same flow rate. The column was washed and eluted as
described for the protein G column.
Monoclonal antibodies 43D1 and 43G8 were purified from cell culture
supernatants by ammonium sulfate precipitation (25) followed
by affinity purification on the acid -glucosidase and protein G
affinity columns, respectively.
Europium labeling of monoclonal antibodies.
Purified
monoclonal antibodies 43D1 and 43G8 were labeled with
Eu3+ chelate, using the
DELFIA® labeling kit (EG&G Wallac), and purified
on a Pharmacia Superose 12 fast-phase liquid chromatography column
(1.5 x 30 cm) as described by Meikle et al. (17). The
coupling efficiencies were determined from protein mass and
fluorescence output of the conjugate.
immunoquantification of acid -glucosidase
Total (precursor and mature) acid -glucosidase protein was
determined with a polyclonal/monoclonal (43D1) sandwich immunoassay,
whereas the mature acid -glucosidase protein was specifically
determined using a monoclonal (43D1)/monoclonal (43G8) sandwich
immunoassay. Each assay was performed as either a one-step assay for
the determination of protein in dried blood spots or a two-step assay
for the determination of protein in plasma samples.
One-step assay.
Microtiter plates (Immulon 4; Dynatech
Technologies) were coated overnight at 4 °C with 100 µL/well of
affinity purified anti-acid -glucosidase polyclonal antibody (2
mg/L) or monoclonal antibody 43D1 (4 mg/L). Coated plates were
prewashed once in DELFIA wash buffer (EG&G Wallac). Dried blood spots
were placed in microtiter wells with 200 µL of assay buffer
containing 200 µg/L of either Eu3+-labeled 43D1
(determination of total -glucosidase) or 43G8 (determination of
mature -glucosidase) monoclonal antibody. The microtiter plates were
shaken (at 20 °C for 60 min) and incubated overnight at 4 °C. The
microtiter plates were again shaken at 20 °C for 60 min, blood-spot
filters were removed by suction, and the plates were washed six times
with DELFIA wash buffer. This was followed by the addition of 200 µL
of DELFIA enhancement solution (EG&G Wallac). The microtiter plates
were shaken at 20 °C for 15 min, and the fluorescence was read on a
DELFIA 1234 Research Fluorometer (EG&G Wallac).
Two-step assay.
Immunoquantification of both the total and the
mature forms of acid -glucosidase in plasma samples collected with
EDTA or citrate as the anticoagulant was performed using the two-step
assay. Plates were coated with antibodies as described for the one-step
assay. Samples were diluted with DELFIA assay buffer (100 µL/well).
The plates were shaken at 20 °C for 60 min and incubated for 5
h at 20 °C. The plates were washed six times, and 100 µL of assay
buffer containing 200 µg/L of either
Eu3+-labeled 43D1 or 43G8 monoclonal antibody was
added to each well. The plates were shaken at 20 °C for 15 min and
incubated overnight at 4 °C. The plates were washed six times, and
DELFIA enhancement solution (200 µL) was added to each well. The
plates were shaken at 20 °C for 15 min, and the fluorescence was
read on a DELFIA 1234 Research Fluorometer.
The concentrations of the total and the mature form of acid
-glucosidase in both the blood spots and the plasma were calculated
using Multicalc Data Analysis software (EG&G Wallac).
preparation of calibrators and quality-control samples
The precursor form of acid -glucosidase was used as a
calibrator for immunoquantification of the total acid -glucosidase,
whereas the mature form was used for the immunoquantification of the
mature protein.
Blood spot calibrators were used to determine the total and mature
forms of acid -glucosidase protein in dried blood spots. The
purified precursor and mature acid -glucosidase proteins were
diluted in buffer (40 g/L human serum albumin, 25 g/L human
-globulin, 20 mmol/L Tris-HCl, 150 mmol/L NaCl, pH 7.8). These
proteins were further diluted threefold with washed sheep red blood
cells to give final concentrations of 200, 100, 50, 25, and 12.5
µg/L. Similarly, two blood spot controls containing low (60 µg/L)
and high (180 µg/L) concentrations of acid -glucosidase protein
were also prepared as described above. Aliquots (50 µL) of the
calibrators and controls were spotted on Whatman 180 BFC filter paper
and were air dried overnight at room temperature.
Liquid calibrators were used to determine the total and mature forms of
acid -glucosidase protein in plasma. Liquid calibrators were
prepared by diluting the precursor and mature forms of acid
-glucosidase protein in DELFIA assay buffer to give final
concentrations of 5, 2.5, 1.25, 0.625, and 0.313 µg/L for the
precursor form and 10, 5, 2.5, 1.25, and 0.625 µg/L for the mature
forms of the protein. Three quality-control samples containing 0.1,
1.0, and 2.0 µg/L precursor protein or 1.0, 2.0, and 8.0 µg/L
mature protein were prepared.
Dried blood spot calibrators and controls were stored in sealed bags containing silica gel at -70 °C. Liquid calibrators and controls were stored at -70 °C. Both dried blood spot and liquid calibrators were assayed in duplicate at the beginning of each plate. Single estimations of the quality-control samples were made for each analytical assay. Dried blood spots were assayed singly, and any repeat samples were assayed in duplicate. All plasma calibrators and samples were assayed in duplicate.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-glucosidase-glucosidase calibrators were >99% pure based on
sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis.
After silver staining, the precursor form showed a single band at 112
kDa, and the mature formed a single band at 81 kDa. These corresponded
to the published precursor (110 kDa) and mature (76 kDa) forms of acid
-glucosidase, respectively (2).
The immunoquantification assay for acid -glucosidase was optimized
by standard procedures to achieve the appropriate assay precision
required for concentrations seen in blood spots and plasma. In
addition, the specificities of the acid -glucosidase antibodies for
the precursor and the mature forms of the protein were determined (Fig. 1
). The degree of cross-reactivity for the precursor form
of the protein by the monoclonal antibody 43G8 was <3% (Fig. 1B
). To
monitor assay performance, three quality-control samples (low, medium,
and high) for plasma and two (low and high) for dried blood spots were
included within each analytical run. Precision profiles for the
analysis of total and mature forms of acid -glucosidase using liquid
calibrators and blood spot calibrators showed CVs <7.5%. Precision
studies for plasma study were conducted over 60 days with 28
observations performed. Blood spot precision studies were conducted
with 36 observations over 30 days. Assays of dried blood spots stored
for different periods of time (1, 4, 12, and 52 weeks) demonstrated
that the dried blood spots were stable for at least 3 months when
stored at room temperature. However, a significant decrease in the
medium concentration (29%) was found after storage for 12 months.
|
acid -glucosidase concentrations in plasma
The total (precursor and mature) and mature forms of acid
-glucosidase protein were determined in plasma samples from 195
control individuals and 404 LSD-affected individuals, representing 26
different disorders (Table 1
and Fig. 2
). In the control population, the total concentration of acid
-glucosidase protein had a skewed distribution with a median of 17.1
µg/L and the 5th and 95th percentiles at 5.6 and 34.7 µg/L,
respectively. The concentration of the mature form of the acid
-glucosidase protein in the control population was very low, with a
median value of 0.2 µg/L and 5th and 95th percentiles at 0 and 3.66
µg/L, respectively. Among the LSD-affected individuals, all
individuals with acid lipase deficiency and 86%, 60%, and 58% of
individuals with mucolipidosis II/III, Niemann-Pick (A/B) disease, and
Gaucher disease, respectively, had total acid -glucosidase
concentrations higher than the 95th percentile of the control group
(Table 1
). In the Pompe disease group, the concentrations of both the
total and mature forms of acid -glucosidase were significantly
reduced (P <0.001) based on nonparametric
statistical analysis (Mann–Whitney test). Only 1 of 22 plasma samples
from Pompe patients had a total acid -glucosidase concentration
within the control range.
|
|
distribution of acid -glucosidase protein in blood spots
The distribution of total acid -glucosidase in blood spots from
the newborn population showed a characteristic skewed distribution
(Fig. 3
). The median concentration of the 1951 blood spots was 59.0
µg/L with 5th and 95th percentiles of 28.6 and 112.0 µg/L,
respectively. The median concentration of the mature form in blood
spots was 53 µg/L with 5th and 95th percentiles of 24 and 110 µg/L,
respectively. The correlation between the total (precursor and mature
forms) and mature forms of the protein in blood spots was 0.83 (Pearson
correlation) and was significant at P = 0.01
(two-tailed). In a separate study, the concentration of total acid
-glucosidase was determined in blood spots from 12 juvenile and 12
adult controls and compared with 20 newborn samples. Nonparametric
statistical analysis (Kruskal–Wallis test) indicated no significant
differences among these groups. The total and mature forms of acid
-glucosidase were measured in dried blood spots from 20 individuals
with Pompe disease and 2 carriers (Table 2
). The concentration of the total acid -glucosidase protein
was lower than the 0.2 percentile of newborn population in 16 of the 17
Pompe patients, whereas the concentration in all carriers tested was
below the 0.6 percentile. In addition, the concentration of both the
total and mature forms of acid -glucosidase protein were measured in
the corresponding plasma from three Pompe patients and one carrier
(Table 2
). The concentrations of the protein were also reduced in the
plasma from these patients, supporting the low values observed in the
dried blood spots.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1 in 5000 births in
Australia) is comparable to other intensively studied genetic disorders
such as cystic fibrosis and phenylketonuria (1 in 2500 and 1 in 14 000
births in Australia, respectively). In most LSDs, the pathology of the
disease is not apparent at birth and manifests in the first few years
of life. If current and proposed therapies are to achieve maximum
efficacy, it will be required that these disorders are detected early,
before the onset of irreversible pathology, particularly central
nervous system and/or bone pathology. Except in cases where a family
history is available, presymptomatic diagnosis of LSDs can be achieved
only by a mass screening program.
In this study, we investigated acid -glucosidase as a potential
screening marker for Pompe disease in particular and for LSDs in
general. Earlier studies showed that LAMP-1 (17) and a
related protein, LAMP-2 (18), were increased in the majority
of LSDs (17) but were not increased in 35% of patients
representing several specific LSDs.
To determine the usefulness of the acid -glucosidase protein as a
screening marker for LSD, we measured the concentrations of both the
total and mature forms of the protein in plasma from LSD-affected
individuals and compared these with concentrations in plasma from
control individuals. The majority of acid -glucosidase in plasma
from the control population was the precursor form. The acid
-glucosidase protein was increased in individuals with acid lipase
deficiency, mucolipidosis II/III, Gaucher disease, and Niemann-Pick
disease (type A/B). The observed increase in plasma from mucolipidosis
II/III patients was identified as the precursor form of the protein.
This results from the mistargeting of newly synthesized protein in
these patients because of the absence of the mannose-6-phosphate moiety
on the enzyme. The increases observed in patients deficient for acid
lipase also represented an increase in the concentration of precursor
protein. However, the increases observed in patients with Gaucher
disease and Niemann-Pick disease (type A/B) resulted primarily from an
increase in the mature form of the protein. This implies a release of
protein from the lysosome in these disorders. This may result from cell
death and subsequent breakdown or alternatively, from specific
exocytosis of lysosomal contents from affected cells. As a general
marker for LSDs, acid -glucosidase has limited application because
it is significantly increased in relatively few disorders.
Plasma samples from 22 patients with Pompe disease showed a significant
decrease in the concentration of the acid -glucosidase protein. Only
one Pompe disease patient had protein concentrations within the control
range. Individuals with significant concentrations of acid
-glucosidase protein may represent different mutations in the gene
that lead to the absence or marked reduction in catalytic capacity of
the mutant enzyme but normal processing (1).
In dried blood spots, 14 of 17 patients had concentrations below the
range of the control population (based on 1951 samples) and 16 of 17
patients were below the 0.4 percentile. One infantile patient had
significant concentrations of acid -glucosidase as determined from
the dried blood spot. The majority of this protein was the mature form
as determined by the assay specific for the mature form, which
indicates lysosomal processing. No mutational data were available on
this patient. A percentage of Pompe patients are reported to have
mutations that lead to significant concentrations of -glucosidase
protein with reduced activity. One such mutation is Asp 645
Glu,
which is reported to be the most common mutation in the Chinese
population in Taiwan, accounting for 36% of mutations (26).
This mutation has been reported to lead to the mistargeting and
misprocessing of the acid -glucosidase protein (27), so
that only the precursor form of the protein is present in the cells.
Clearly this is not the mutation present in our high-protein patients.
However, a potential approach to the identification of patients
expressing only the precursor form is to screen for the mature form of
the acid -glucosidase protein. This approach would be limited to
whole blood or blood spot samples because the concentration of mature
protein in plasma is low in the unaffected population.
We have demonstrated that it is feasible to reliably measure
the total acid -glucosidase protein (precursor and mature forms) and
the mature form only in either dried blood spots or plasma. In
addition, we have shown a good correlation between the absence or
reduced concentrations of total acid -glucosidase protein and the
incidence of Pompe disease. For plasma samples, when we used a cutoff
concentration of 2.0 µg/L, the total acid -glucosidase assay gave
a sensitivity of 95% with a specificity of 100% (21 of 22 patient
samples were below the control range based on 195 unaffected,
age-matched control subjects). Analysis of the blood spot samples using
a cutoff concentration of 6 µg/L gave a sensitivity of 82% with a
specificity of 100% (14 of 17 patients were below the range of the
control population). Increasing the cutoff to 11 µg/L increased the
sensitivity (94%) but decreased the specificity (99.8%). A
specificity of 99.8% would produce a large number of false positives
that may also include many carriers, although the numbers of carriers
in this study are insufficient to determine this value accurately.
Although these studies were based on a comparison of infantile,
juvenile, and adult patients with a control population of 1951
newborns, comparison of smaller control groups of newborns, juveniles,
and adults showed no statistically significant difference in the
concentration of acid -glucosidase, indicating no age correlation.
On the basis of this study, it is feasible to screen for Pompe disease
by determining the concentration of total acid -glucosidase protein
in plasma or dried blood spots. However, additional work needs to be
done to increase the sensitivity without a corresponding decrease in
the specificity. One possible approach is the development of a
two-tiered screening strategy involving an initial protein
determination followed by an enzyme activity determination made on a
second blood spot from the same Guthrie card. The second-tier assay
would be performed on the top 0.5% of the population as determined
from the first-tier assay. Alternative approaches may involve mutation
analysis or determination of lysosomal substrate storage as a second
tier of the screen. Additional work in these areas is ongoing.
|
Acknowledgments |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-glucosidase (acid maltase) deficiency. Scriver CR Beaudet AC Sly WS Valle D eds. The metabolic and molecular bases of inherited disease, 7th ed 1995;Vol. II:2443-2464 McGraw-Hill New York. .
-glucosidase during intracellular transport and maturation. J Biol Chem 1993;268:2223-2231.-glucosidase from rabbit milk has therapeutic effect in mice with glycogen storage disease type II. Hum Mol Genet 1999;8:2145-2153.-glucosidase gene into fibroblasts, myoblasts and myotubes from patients with glycogen storage disease type II leads to high level expression of enzyme and corrects glycogen accumulation. Hum Mol Genet 1998;7:1695-1702.-glucosidase deficiency in Pompe disease fibroblasts in vitro, and lysosomally targeted expression in neonatal rat cardiac and skeletal muscle. Gene Ther 1998;5:473-480.[Web of Science][Medline]
[Order article via Infotrieve]
-glucosidase cDNA to enzyme-deficient myoblasts results in phenotypic spread of the genotypic correction by both secretion and fusion. Hum Gene Ther 1997;8:1555-1563.[Web of Science][Medline]
[Order article via Infotrieve]
-glucosidase. Proc Natl Acad Sci U S A 1999;96:8861-8866.-D-glucosidase for study of Chinese glycogenesis type II patients. Hybridoma 1992;11:493-505.[Web of Science][Medline]
[Order article via Infotrieve]
-glucosidase. Eur J Biochem 1984;139:497-502.[Web of Science][Medline]
[Order article via Infotrieve]
-glucosidase affects transport and phosphorylation of the enzyme in an adult patient with glycogen-storage disease type II. Biochem J 1993;289:687-693.
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  Mohd. A. F. Tan, C. J. Dean, J. J. Hopwood, and P. J. Meikle Diagnosis of Metachromatic Leukodystrophy by Immune Quantification of Arylsulphatase A Protein and Activity in Dried Blood Spots Clin. Chem., November 1, 2008; 54(11): 1925 - 1927. [Full Text] [PDF]
|
|
|
|
 |
|
 A. R. Kemper, W.-L. Hwu, M. Lloyd-Puryear, and P. S. Kishnani Newborn Screening for Pompe Disease: Synthesis of the Evidence and Development of Screening Recommendations Pediatrics, November 1, 2007; 120(5): e1327 - e1334. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. E. Sleat, Y. Wang, I. Sohar, H. Lackland, Y. Li, H. Li, H. Zheng, and P. Lobel Identification and Validation of Mannose 6-Phosphate Glycoproteins in Human Plasma Reveal a Wide Range of Lysosomal and Non-lysosomal Proteins Mol. Cell. Proteomics, October 1, 2006; 5(10): 1942 - 1956. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Parkinson-Lawrence, M. Fuller, J. J. Hopwood, P. J. Meikle, and D. A. Brooks Immunochemistry of Lysosomal Storage Disorders Clin. Chem., September 1, 2006; 52(9): 1660 - 1668. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Fuller, M. Lovejoy, J. J. Hopwood, and P. J. Meikle Immunoquantification of {beta}-Glucosidase: Diagnosis and Prediction of Severity in Gaucher Disease Clin. Chem., November 1, 2005; 51(11): 2200 - 2202. [Full Text] [PDF]
|
|
|
|
|
|
P. J. Meikle, E. Ranieri, H. Simonsen, T. Rozaklis, S. L. Ramsay, P. D. Whitfield, M. Fuller, E. Christensen, F. Skovby, and J. J. Hopwood Newborn Screening for Lysosomal Storage Disorders: Clinical Evaluation of a Two-Tier Strategy Pediatrics, October 1, 2004; 114(4): 909 - 916. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Rozaklis, S. L. Ramsay, P. D. Whitfield, E. Ranieri, J. J. Hopwood, and P. J. Meikle Determination of Oligosaccharides in Pompe Disease by Electrospray Ionization Tandem Mass Spectrometry Clin. Chem., January 1, 2002; 48(1): 131 - 139. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Raben, N. Lu, K. Nagaraju, Y. Rivera, A. Lee, B. Yan, B. Byrne, P.J. Meikle, K. Umapathysivam, J.J. Hopwood, et al. Conditional tissue-specific expression of the acid {alpha}-glucosidase (GAA) gene in the GAA knockout mice: implications for therapy Hum. Mol. Genet., September 1, 2001; 10(19): 2039 - 2047. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Umapathysivam, J. J. Hopwood, and P. J. Meikle Determination of Acid {alpha}-Glucosidase Activity in Blood Spots as a Diagnostic Test for Pompe Disease Clin. Chem., August 1, 2001; 47(8): 1378 - 1383. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and
mature (
) forms of the protein were assayed for total protein
(A) using monoclonal antibody 43D1 and for mature
protein (B) using monoclonal antibody 43G8 as described
in Materials and Methods. Under standard assay
conditions, a linear response was observed over the range 0–20 µg/L
for both the precursor and mature forms of acid 
