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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 14, 2004 as doi:10.1096/fj.03-0941fje. |
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Department of Genetics and Tumor Cell Biology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA
2Correspondence: Department of Genetics and Tumor Cell Biology, Mail Stop 331, St. Jude Children’s Research Hospital, 332 N. Lauderdale, Memphis, TN 38105-2794. E-mail: alessandra.dazzo{at}stjude.org
SPECIFIC AIMS
Lysosomal storage diseases (LSDs) are monogenic disorders of metabolism caused by deficiency of hydrolytic enzymes. Enzyme replacement therapy (ERT) is commercially available for some groups of patients with non-neuropathic forms of the disease, but most LSDs remain untreated. LSDs such as galactosialidosis (GS) in which the reticuloendothelial (RE) system is primarily involved could be treated efficiently by targeting the therapeutic enzyme exclusively to RE cells via the mannose receptor. Baculovirus-mediated production of recombinant enzymes in insect cells might be suitable and advantageous, because glycoproteins expressed in these cells carry mannosylated N-glycans and are poised to be internalized by cells that express the mannose receptor. GS is caused by a primary defect of the carboxypeptidase protective protein/cathepsin A (PPCA), but disease symptoms are primarily the result of a severe secondary deficiency of lysosomal neuraminidase (NEU1). The aim of this study was to determine which cells and tissues of the mouse model of GS internalize insect cell-produced PPCA and Neu1 and whether this ERT corrects enzyme deficiency and reverses or prevents lysosomal vacuolization.
PRINCIPAL FINDINGS
1. Insect cell-expressed PPCA and Neu1 are efficiently internalized by mouse GS macrophages and restore catalytic activity
GS mouse macrophages were cultured in the presence of either PPCA alone or a combination of PPCA and Neu1. After 4 h, the enzyme-containing medium was replaced with enzyme-free medium and cells were harvested at different times (0, 2, 18, 24, and 42 h) after uptake. At t = 0, the uptake of PPCA alone restored the cathepsin A (CA) activity to fivefold that of normal cells. CA activity decreased to twofold that of normal cells after 42 h (Fig. 1A
). The uptake of PPCA also rescued endogenous Neu1 activity, which gradually increased during the first 24 h to 145% of the activity in normal macrophages. The combined uptake of PPCA and Neu1 restored the CA activity to 
50% of the values measured after uptake of PPCA alone. This could be due to competition between the two enzymes for the mannose receptors on macrophages. However, the rate of decrease in CA activity over time was similar to that observed after uptake of PPCA alone. Neu1 activity was substantially higher after the combined uptake of PPCA and Neu1, although it declined more rapidly than PPCA. Administration of PPCA and Neu1 in the presence of mannan, a competitive inhibitor of the mannose receptor, inhibited uptake of the enzymes, as evidenced by a negligible increase in enzyme activity. Western blot analysis of macrophage lysates using anti-PPCA and anti-Neu1 antibodies confirmed a gradual decrease of internalized Neu1 and PPCA. Again, the decrease of Neu1 protein was more rapid than that of PPCA.
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2. Insect cell-expressed PPCA and Neu1 are internalized by macrophages via the mannose receptor but not via the mannose-6-phosphate receptor, the common receptor for lysosomal enzymes
After uptake of PPCA and Neu1, immunofluorescence of GS macrophages using anti-PPCA and anti-Neu1 antibodies showed a punctuated intracellular distribution of both enzymes, a finding consistent with lysosomal localization. Mannose-6-phosphate, a competitive inhibitor of the M6P receptor, did not affect the amount and intracellular distribution of PPCA or Neu1. In contrast, mannan inhibited endocytosis of the recombinant enzymes as demonstrated by the significantly reduced amount of enzymes detected in the cells. Thus, PPCA and Neu1 were readily internalized by macrophages exclusively via the mannose receptor and correctly transported to lysosomes.
3. PPCA and Neu1 are detected in the systemic organs of GS mice after intravenous administration of recombinant enzymes
The efficient uptake of PPCA and Neu1 by enzyme-deficient macrophages and restoration of the catalytic activities suggested that these baculovirus-expressed enzymes could be suitable for ERT in GS mice. To correct PPCA and Neu1 deficiencies in visceral organs and to maximize the reduction of lysosomal vacuolization, we tested i.v. administration of either PPCA alone or PPCA and Neu1. We first established the optimal frequency of administration and the duration of detection of the enzymes in the visceral organs after injection. Because the half-life of PPCA (42 h) is longer than that of Neu1 (30 h), we administered 150 µg PPCA and 300 µg Neu1. The enzymes were injected i.v. in 1-month-old GS mice. Treated mice were killed 4, 24, 48, 72, and 96 h after injection, and tissues were analyzed using immunohistochemical and Western blot methods with anti-PPCA and anti-Neu1 antibodies. PPCA and Neu1 were abundant in resident macrophages of the liver and spleen 4 and 24 h after injection. Although immunolabeling was progressively weaker in tissues of mice killed later, PPCA was still detected for up to 96 h and Neu1 for up to 72 h after injection. The amount of PPCA detected in mice injected with PPCA alone did not differ from that seen in mice injected with a combination of PPCA and Neu1. These results indicate that for ERT of GS mice, administration once every 2 to 3 days of PPCA alone or a combination of PPCA and Neu1 should be sufficient.
4. Lysosomal storage and vacuolization was reduced and overall tissue morphology was improved in GS mice treated with insect cell-expressed PPCA and Neu1
We initiated ERT on 1-month-old GS mice by injecting three times/wk either PPCA alone (mono-ERT) or a combination of Neu1 and PPCA (dual ERT). After 2 wk of mono-ERT or dual ERT, some systemic organs like the liver, spleen, and bone marrow showed complete restoration of CA activity; others, like the kidneys, lungs, and gonads, had partial correction. Neu1 activity was completely restored in the visceral organs of only those mice that received dual ERT and partially restored in the liver of mice that received mono-ERT. We did not detect CA or Neu1 activity in the brain of treated mice.
Western blot analysis with anti-PPCA antibodies confirmed the presence of the protein in the liver, spleen, bone marrow, and kidney of mice from both treatment groups. Immunohistochemical analysis showed an abundance of PPCA in the Kupffer cells of the liver, macrophages in the red pulp of the spleen, and macrophages in the kidney; abundant immunolabeling was detected in tissues from both treatment groups. PPCA-positive macrophages were seen, albeit more sporadically, in other organs including heart, adrenal gland, intestine, testis, and choroid plexus. Immunostaining of Neu1 in tissues of GS mice that received dual ERT closely paralleled the levels of enzyme activity: Neu1 was readily detected in Kupffer cells of the liver, macrophages of the spleen, and the Bowman capsule of the kidney.
Livers of untreated GS mice contained twice the amount of sialic acid than that present in wild-type samples, indicating excessive storage of sialoglycoconjugates. The livers of mono- and dual ERT-treated mice both showed a decrease in the amount of sialic acids to the level detected in wild-type mice. Therefore, mono- and dual ERT were equally efficient in reducing lysosomal storage in the liver of GS mice, confirming the efficacy of the treatment in clearing lysosomal storage products.
Consistent with biochemical findings, hematoxylin and eosin (H&E) staining of tissues from GS mice that received dual ERT showed a substantial reduction in vacuolization, and overall tissue morphology was dramatically improved in the organs most affected by the disease (i.e., liver, kidney, and spleen) (Fig. 1
). In contrast, the kidney and spleen from GS mice that received mono-ERT were significantly vacuolated (Fig. 1 K, L
). Thus, the combined uptake of PPCA and Neu1 (dual ERT) by macrophages appears to be most effective in reducing lysosomal storage in visceral organs of GS mice. Even tissues that showed only a marginal correction of enzyme activity (e.g., kidney) showed dramatically reduced vacuolization and improved tissue morphology.
CONCLUSIONS AND SIGNIFICANCE
The composition of the N-glycans of a lysosomal enzyme determines which cell types are possible targets for the protein’s uptake and, in turn, its suitability for ERT. Most soluble lysosomal hydrolases expressed in mammalian cells carry the M6P recognition marker and can be internalized by various cell types via the M6P receptor. In contrast, insect cell-expressed glycoproteins expose mainly core-type pauci-mannose carbohydrates that render them suitable for uptake by RE cells via the mannose receptor. Several lysosomal hydrolases, including Neu1, lack a functional M6P recognition marker; thus, expression in insect cells and uptake via the mannose receptor in mammalian cells could circumvent this problem and be the sole method of enzyme production for ERT.
It is unclear whether overall morphologic improvements in the systemic organs of treated GS mice are the result of a correction of only RE cells. This can best be illustrated by changes seen in the liver after ERT. It is known that liver can undergo cycles of hepatocyte regeneration after acute or subacute injury. Newly divided hepatocytes can be distinguished from polyploid resting cells by the histologic appearance of their nuclei, which are small and uniform. In our study, the livers of untreated and treated GS mice both contained many newly divided hepatocytes, which could reflect tissue regeneration. We can hypothesize that injury caused by extensive lysosomal vacuolization also induces hepatocyte regeneration. This process is driven by multipotent liver stem cells in the terminal biliary ductules that proliferate into oval cells, which in turn differentiate into hepatocytes. Hematopoietic stem cells in the bone marrow have been implicated in the proliferation of oval cells and hepatocytes in the pancreas and extrahepatic bile ducts. Tumor necrosis factor stimulates Kupffer cells to secrete interleukin-6, which initiates the regeneration of hepatocytes. Thus, by correcting the enzyme deficiency in Kupffer cells, we may enhance their ability to promote hepatocyte regeneration. Similar processes may take place in other organs.
Our studies suggest that ERT with BV-expressed PPCA and NEU1 may be beneficial to juvenile patients who have GS without CNS involvement and for those with clinically similar type II sialidosis. The cells of patients who have juvenile GS or type II sialidosis express residual amounts of PPCA and NEU1; therefore, the chances of adverse immunologic reactions toward the therapeutic proteins may be minimal. ERT is used to treat only a few LSDs because of the relatively low incidence of each disorder and the inevitably high costs associated with the development and production of therapeutic proteins. Many LSDs, however, are genetically heterogeneous, and patients with non-neuropathic forms may benefit from ERT. Production of enzymes in insect cells could be an alternative source of therapeutic enzyme for LSDs like GS, because insect cell-produced enzymes would eliminate the need for treatment with exoglycosidases to expose mannose residues. Application of insect cells for production of therapeutic proteins could eventually widen the availability of ERT for patients with non-neuropathic forms of other LSDs.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0941fje; 
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