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1
Lysosomal Diseases Research Unit, Department of Chemical Pathology, Women’s and Children’s Hospital, 72 King William Road, North Adelaide, South Australia 5006, Australia.
2
School of Biological Sciences and Medicine, The Flinders
University of South Australia, P.O. Box 2100, Adelaide 5001, Australia.
3
Division of Human Genetics, Children’s Hospital Medical
Center, 3333 Burnet Ave., Cincinnati, OH 45229.
4
Biochemistry, Endocrinology and Metabolism Unit,
Institute of Child Health (University College London), 30 Guilford St.,
London WC1N 1EH, UK.
a Author for correspondence. Fax 61-8-204-7100; e-mail pmeikle{at}medicine.adelaide.edu.au
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: Four time-delayed fluorescence immunoquantification assays were used to measure each of the saposins in plasma from 111 unaffected individuals and 334 LSD-affected individuals, representing 28 different disorders.
Results: Saposin A was increased above the 95th centile of the control population in 59% of LSD patients; saposins B, C, and D were increased in 25%, 61%, and 57%, respectively. Saposins were increased in patients from several LSD groups that in previous studies did not show an increase of lysosome-associated membrane protein-1 (LAMP-1).
Conclusion: Saposins may be useful markers for LSDs when used in conjunction with LAMP-1.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Predominantly affecting young children, LSDs can present with a wide range of clinical symptoms that depend on the genotype involved, including mental retardation, skeletal abnormalities, organomegaly, corneal clouding, and coarse facial features (1)(2). In recent years, treatments for several LSDs have become possible, e.g., cystinosis is treated with cysteamine (3)(4), several LSDs have been responsive to bone marrow transplants (5)(6)(7), and Gaucher disease currently is being treated by enzyme replacement therapy (8), which like bone marrow transplantation, is theoretically applicable to a wide range of LSDs (9). Studies in animal models have shown that maximum efficacy, in most cases, is achieved when treatment is given at an early stage of pathology (10)(11)(12).
Most individuals are brought to attention only after the presentation of clinical symptoms. The diagnosis of most LSD-affected individuals is carried out using a range of enzymatic assays, performed on urine, blood, and skin fibroblasts. These assays are time-consuming, costly, and invasive. The possibility of newborn screening for LSDs has been discussed (13), and a strategy with a two-tier system has been proposed (14).
For newborn screening for LSDs to be economically viable, screening markers are required that will enable the detection of most LSDs in a single procedure. In a previous study, the plasma concentrations of lysosome-associated membrane protein-1 (LAMP-1) were above the 95th centile of the control population in 72% of LSD-affected individuals (13). Because the remaining LSDs not identified by LAMP-1 were predominantly those that stored sphingolipids, we have studied the sphingolipid activator proteins, or saposins, as potential complementary markers.
The saposins are four small, heat-stable glycoproteins, termed saposins A, B, C, and D; all are derived from a single 73-kDa precursor protein, called prosaposin. The mature saposins have specific roles in activating and enhancing the activities of their respective lysosomal hydrolases (15)(16). Saposins are critical in the control of the glycosphingolipid flux through the lysosomal hydrolytic pathway (16), and genetic defects in sphingolipid hydrolases and/or saposins have been associated with the storage of sphingolipids (17). Conversely, saposin accumulations have been reported in the tissues of several LSD-affected individuals. Increases as much as 80-fold have been observed in the spleens, livers, and brains of patients affected with Gaucher disease, Niemann-Pick disease (type 1), fucosidosis, Tay-Sachs disease, and Sandhoff disease (18)(19).
In this study, we evaluated the frequency of increases in saposins A, B, C, and D in LSDs by measuring the concentrations of these proteins in plasma samples taken from unaffected and LSD-affected individuals.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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polyclonal antibodies
Polyclonal antibodies against saposins A, B, C, and D were
produced and characterized as described previously (16).
Each antibody was purified on a 2-mL HitrapTM
Protein G column (Pharmacia Biotech) and quantified by absorbance at
280 nm (absorbance = 1.4 for 1.0 g/L).
europium labeling of polyclonal antibodies
Purified anti-saposin polyclonal antibodies were labeled with
Eu3+, using the DELFIA®
labeling kit (Wallac). Labeled antibodies were purified on a 1.5
x 30 cm Superose 12 fast-phase liquid chromatography column (Pharmacia
Biotech) as described previously (13). The amount of
Eu3+ conjugated to each antibody molecule was
determined by the fluorescence of a known antibody concentration
compared with a 1 nmol/L Eu3+ solution.
preparation of calibrators and quality-control materials
Liquid calibrators for the immunoquantification of saposins were
prepared using purified saposin A, B, C, and D proteins
(21). Purified saposin A protein was diluted in DELFIA assay
buffer to obtain final concentrations of 2.43, 1.21, 0.61, 0.3, and
0.15 µg/L. For the saposin A immunoquantification assays, low and
high quality-control samples were prepared by diluting saposin A
protein to 0.24 and 2.43 µg/L, respectively. Purified saposin B
protein was diluted in the same buffer to obtain final concentrations
of 6.68, 3.34, 1.67, 0.83, and 0.42 µg/L. The saposin B low and high
quality-control samples were prepared by diluting saposin B protein to
0.67 and 6.68 µg/L, respectively. Purified saposin C protein was
diluted to obtain final concentrations of 1.88, 0.94, 0.47, and 0.24
µg/L. The saposin C low and high quality-control samples were
prepared by diluting saposin C protein to 0.47 and 1.88 µg/L,
respectively. Purified saposin D protein was diluted to obtain final
concentrations of 0.92, 0.46, 0.23, and 0.11 µg/L. The saposin D low
and high quality-control samples were prepared by diluting saposin D
protein to 0.23 and 0.92 µg/L, respectively.
Calibrators and controls for saposins C and D were supplemented with a volume of control plasma equal to that being assayed to compensate for the inhibitory effects of plasma on the saposin C and D assays. All liquid saposin calibrators and controls were stored at 4 °C.
immunoquantification of saposins
Saposin concentrations were determined by time-delayed
fluorescence immunoassays as described previously (13).
Briefly, microtiter plates (Immulon 4; Dynatech Laboratories) were
coated overnight at 4 °C with anti-saposin polyclonal antibody (2.5
mg/L diluted in 0.1 mol/L NaHCO3, pH 8.8; 100
µL/well), and then prewashed once with DELFIA wash buffer. Samples
were diluted in DELFIA assay buffer (100 µL/well), shaken for 10 min
at 20 °C, and incubated (4 h at 4 °C). Plates were then washed
six times, and Eu3+-labeled anti-saposin
polyclonal antibody added (0.25 mg/L diluted in DELFIA assay buffer;
100 µL/well) and incubated overnight at 4 °C. The plates were
washed six times; DELFIA enhancement solution (200 µL) was then added
to each well, and the plates were shaken (10 min at 20 °C). The
fluorescence was read on a Wallac 1234 DELFIA Research Fluorometer. For
each saposin assay, the corresponding saposin calibrators were located
across the first row of the microtiter plate, with the quality-control
samples dispersed randomly. All saposin calibrators, quality controls,
and samples were assayed in duplicates. Saposin concentrations in
plasma samples were calculated using linear regression.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The optimized saposin A, B, and C calibration curves gave linear
responses over the range of the calibrators
(R2 values >0.99), with the exception
of the saposin D calibration curve, which had a reduced linear range
(R2 = 0.9575; Fig. 1
). When control plasma was added to the saposin C and D
calibration curves, a reduction in signal intensity was observed, 2
µL of plasma gave a 20% reduction (data not shown). To compensate
for the inhibitory effect of plasma on the saposin C and D
immunoquantification assays, control plasma at an equal volume to that
being assayed was added to the calibrators. The assumption was made
that LSD-affected plasma would inhibit the assays in the same manner as
control plasma.
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We evaluated the cross-reactivity between the saposin assays and found
that it was <3% for the saposin A, B, and C assays, whereas the
saposin D assay showed between 6% and 13% cross-reactivity with the
other saposins (Table 1
).
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saposin concentrations in the plasma of control and lsd-affected
individuals
To evaluate the suitability of each of the saposins as screening
markers for LSD, the concentrations of saposins A, B, C, and D were
measured in the plasma samples from 111 control individuals (median
age, 7 years; range, 0–66 years) and 334 LSD-affected individuals,
representing 28 different disorders (Table 2
). The 95th centiles of saposin concentrations in the control
population are listed in Table 2
, together with the total proportion of
LSD-affected individuals having plasma saposin concentrations above
this value. Saposins A, C, and D showed a tight distribution in the
plasma samples of the control population, whereas a wider range of
saposin B was observed (Fig. 2
). A significant proportion of LSD-affected individuals were
found to have saposin concentrations above the 95th centile of the
control population, with some individuals having increases of up to
10-fold above the median concentration of the control population. When
the disorders were taken separately, it was noted that in 15 of the 28
disorders, >80% of individuals had one or more saposins above the
95th centile of the control population.
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All saposin assays included low and high quality-control samples to
determine the interassay coefficient of variation (CV) for each assay
performed (Table 3
), with the number of replicates ranging from 17 to 24. The
interassay CVs of the saposin A assays for both the low and high
quality-control samples were 
11%. The saposin B, C, and D assays had
CVs 10% for the high quality-control samples and CVs of 11–20% for
the low quality-control samples. A separate experiment was carried out
in which 24 replicate samples of the high and low quality-control
samples were assayed on a single plate to calculate the intraassay CV
(Table 3
). The intraassay CVs were 16% for the low quality-control
samples and 10% for the high quality-control samples.
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Because the LAMP-1 concentrations in the same plasma samples had been
assayed previously (13), direct comparisons between the
saposin and LAMP-1 concentrations could be carried out. Strong Pearson
correlations were observed between all saposins (P <0.01),
with the exception of saposin B, which showed low correlation with both
saposin C and D (Table 4
). Weak correlations were observed between the saposins and
LAMP-1 (P <0.05), whereas no correlations were seen between
saposin concentrations and age, in the control population. The
correlation between age and the saposin concentrations was also
examined in Fabry disease, Gaucher disease, metachromatic
leukodystrophy (MLD), mucopolysaccharidosis (MPS) II, and Tay-Sachs
disease, where there were >20 patients in each group. Of these groups,
only the Gaucher disease group showed a slight but significant
(P <0.05) negative correlation between age and saposin C
and D concentrations (Pearson correlation of -0.39 and -0.35,
respectively).
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saposin concentrations in whole blood
Whole-blood samples from six unaffected individuals were
fractionated, and the distribution of saposins was determined in
plasma, white cells, and red cells (Table 5
). It was found that the dominant saposin in whole blood was
saposin D, at a concentration of 75 µg/L, whereas a greater
proportion of saposin B (77%) was found in the plasma fraction
compared with the other saposins. The distributions of saposins A, C,
and D in plasma, white cells, and red cells were approximately equal.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The saposin D calibration curve (Fig. 1D
) had a reduced linear range
and low signal compared with the other saposin calibration curves (Fig. 1
, A–C), indicating that the anti-saposin D polyclonal antibody had a
relatively low affinity. The higher affinity of the anti-saposin A
antibody was reflected in the lower inter- and intraassay CVs obtained
for the saposin A assay (Table 3
), where the CV values were 11% for
both the high and low quality-control samples. The intra- and
inter-assay CVs of saposin B for the low quality-control samples were
11% and 20%, respectively, reflecting the lower affinities of these
antibodies. Upon investigation of the cross-reactivity of the
antibodies to the other saposin proteins, we found that the polyclonal
antibodies were of high specificity for their respective saposins with
the exception of saposin D, which had a slight cross-reactivity to the
other saposins (Table 1
).
In a previous study, plasma caused a reduction in the fluorescent signal in the LAMP-2 immunoquantification assay (14). We therefore investigated this effect for saposins and observed a slight reduction in signal for the saposin C and D assays. Three possibilities involving plasma proteins may contribute to this inhibitory effect. First, proteins present in the plasma may bind to saposins C and D, altering their conformation and rendering them unrecognizable to their antibodies. Second, plasma proteins may competitively occupy the saposin-binding sites of the antibodies, inhibiting them from binding the saposins. Third, plasma proteins may bind and aggregate the saposins or their antibodies, thus reducing the signals obtained.
The goal of this study was to identify additional protein markers for
use in a newborn-screening program for LSDs. However, the lack of
suitable diagnostic samples from newborns with LSDs necessitated the
use of plasma samples from LSD-affected individuals taken at the time
of diagnosis. The age of patients at diagnosis varied considerably with
disorder (Table 2
) and was generally <10 years of age. The control
population was age-matched to the LSD-affected group. Comparison of
fresh control samples with those stored frozen for many years showed no
difference in the median values, indicating the stability of the
saposins in these samples.
We measured the saposin concentrations in plasma from LSD-affected
individuals and compared these to values in plasma from control
individuals. In 59% of patients, saposin A was increased above the
95th centile of the control population, and saposins B, C, and D were
increased in 25%, 61% and 57% of patients, respectively (Table 2
).
Of the 28 disorders represented in our study, the saposin A
concentrations were above the 95th centile of the control group in
>80% of individuals in 6 disorders, saposin B concentrations were
increased in 2 disorders, and saposins C and D were increased in 10
disorders (Table 2
). Together the saposins identified >80% of
individuals in 15 of the 28 LSD groups; increased LAMP-1 concentrations
had not been observed previously in 6 of these LSDs
(13), namely cystinosis, Fabry disease, Niemann-Pick disease
(types A/B and C), Pompe disease, and Wolman disease. The remaining
LSDs in which LAMP-1 was not increased (Krabbe disease, MLD, and
Tay-Sachs disease) had significant numbers of patients (44–71%) with
increases in saposin concentrations (Table 2
). We calculate that up to
85% of LSD-affected individuals can be detected using a combination of
LAMP-1 and saposins as screening markers.
It is worth noting that 13 of the LSD plasma samples were from newborns (<6 weeks of age), representing Gaucher (n = 3), Krabbe (n = 1), MLD (n = 2), MPS VI (n = 2), mucolipidosis II/III (n = 1), Niemann-Pick C (n = 2), Pompe (n = 1), and Tay-Sachs (n = 1) disorders. Of these, 11 (85%) showed an increase in one or more of the saposins above the 95th percentile of the control population, Tay-Sachs and Krabbe disorders being the exceptions.
The ratios of individual saposin concentrations in the plasma from the
LSD group were also examined and compared with the control population
to determine whether the ratio value was a better marker for LSD
detection. However, because the correlations between the saposins were
strong (Table 4
), the ratios of any two saposins gave a lower
predictive value than the individual saposins.
Theoretically, because saposins originate from the same precursor,
their rate of synthesis should be equal. In this study, saposin B was
by far the predominant saposin in the plasma of control individuals
(Table 2
). Analysis of the distribution of saposins in fractions of
whole blood (Table 5
) revealed that saposin D was the most abundant
saposin in whole blood, at 75 µg/L. A greater proportion of saposin B
(77%) was found in the plasma fraction compared with saposins A, C,
and D (38%, 23%, and 30%, respectively), which may explain the high
concentrations observed in the plasma samples in this study
Studies on the distribution of saposins in tissue samples from LSD patients and control individuals also found that saposin D is more abundant in unaffected tissue than the other saposins (22). This observation together with our findings suggests that saposin D may have a longer half-life in cells. The higher concentrations of saposin B in plasma compared with whole blood may suggest a functional difference in extracellular and intracellular saposins and could result from either an increased half-life of this saposin in plasma or its preferential release from cells. However, the mechanism by which saposins are released into circulation is unclear.
Three hypotheses previously have been suggested for the accumulation of saposins in tissues of LSD patients, which may also apply to their increase in plasma: (a) The synthesis of saposins may be stimulated by the accumulation of either the defective enzyme or lipid substrates as a compensatory mechanism. (b) Saposins may be codeposited with the accumulated substrates. (c) Saposins may not be able to interact with deficient enzymes and therefore cannot undergo normal metabolism (23). However, other studies have shown that saposin accumulation may be nonspecific in that it is unrelated to the catabolic defect (15). Because saposin B is known to stimulate the hydrolysis of cerebroside sulfate, the storage product of MLD, we expected that it would be increased in plasma from MLD-affected individuals; however, this was not the case. This observation would suggest that saposin B does not accumulate with its substrate in MLD, although this may be a tissue-specific phenomenon that is not reflected in plasma concentrations of saposin B.
We also observed that there was a general correlation between the LSD
groups that store sphingolipids or sphingolipid derivatives and those
that showed an increase in saposin concentrations. For example,
Niemann-Pick disease (type A/B), in which sphingomyelin is stored,
showed increases in all saposins, whereas of the MPS groups studied,
only MPS II showed an increase in saposin C (Table 2
). In addition,
saposins were not increased to the same extent in demyelinating
disorders such as MLD and Krabbe disease, compared with other
lipidoses. This has also been noted in previous tissue studies
(22). The observed increase of saposins in the various LSD
groups may therefore relate to both the tissues involved in storage and
the type of substrate stored. Moreover, the degree of saposin increase
may also reflect the severity of the disorder.
The results presented in this study are based on the analysis of plasma samples from clinically affected LSD patients varying in age from newborns to adults. Although LSDs are progressive disorders that often take years to present clinically, there is considerable evidence from both human (24)(25) and animal models (11)(26) that biochemical storage commences early in gestation and is well advanced at birth. The presence of increased saposin concentrations in 11 of the 13 newborns in the LSD group would also suggest that, at least in these individuals, the biochemistry is well advanced at birth. Although these results support the usefulness of saposins as screening markers for LSD, further studies on the newborn population will be required to confirm the usefulness of saposins as markers for the early detection of LSD.
As with the LAMP-1 and LAMP-2 studies (13)(14), we have used the 95th pecentile as a cutoff for the control population; however, a 5% recall would clearly be unacceptable in a newborn-screening program. We propose to develop a two-tier screening program in which the top 1–5% of the population (based on the primary screening markers) would then be examined further. Using the same Guthrie card, a panel of second-tier diagnostic assays designed to detect the storage product for the particular disorder involved would be performed. This would effectively identify the affected individuals from the false positives identified in the first-tier screen. We currently are developing the second-tier assays for this procedure, utilizing tandem mass spectrometry.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-L-iduronidase replacement
therapy in mucopolysaccharidosis I: results of a human clinical trial.
The American Society of Human Genetics 48th Annual Meeting, October
27–31, 1998, Denver, Colorado:128..
The following articles in journals at HighWire Press have cited this article:
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  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]
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C. J. Dean, M. R. Bockmann, J. J. Hopwood, D. A. Brooks, and P. J. Meikle Detection of Mucopolysaccharidosis Type II by Measurement of Iduronate-2-Sulfatase in Dried Blood Spots and Plasma Samples Clin. Chem., April 1, 2006; 52(4): 643 - 649. [Abstract] [Full Text] [PDF]
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M. Fuller, M. Lovejoy, D. A. Brooks, M. L. Harkin, J. J. Hopwood, and P. J. Meikle Immunoquantification of {alpha}-Galactosidase: Evaluation for the Diagnosis of Fabry Disease Clin. Chem., November 1, 2004; 50(11): 1979 - 1985. [Abstract] [Full Text] [PDF]
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 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]
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 D. A. Wenger, S. Coppola, and S.-L. Liu Insights Into the Diagnosis and Treatment of Lysosomal Storage Diseases Arch Neurol, March 1, 2003; 60(3): 322 - 328. [Abstract] [Full Text] [PDF]
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 J. Piatigorsky, B. Norman, L. J. Dishaw, L. Kos, J. Horwitz, P. J. Steinbach, and Z. Kozmik J3-crystallin of the jellyfish lens: Similarity to saposins PNAS, October 23, 2001; 98(22): 12362 - 12367. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
denotes
the outliers; * denotes extreme outliers. GM I, GM I
gangliosidosis; mucolipidosis, mucolipidosis type
II/III; Mann,