Mannose 6-Phosphate Receptor-mediated Uptake Is Defective in Acid Sphingomyelinase-deficient Macrophages

Abstract

Progressive accumulation of lipid-laden macrophages is a hallmark of the acid sphingomyelinase (ASM)-deficient forms of Niemann-Pick disease (i.e. Types A and B NPD). To investigate the mechanisms underlying enzyme replacement therapy for this disorder, we studied the uptake of recombinant, human ASM (rhASM) by alveolar macrophages from ASM knock-out (ASMKO) mice. The recombinant enzyme used for these studies was produced in Chinese hamster ovary cells and contained complex type, N-linked oligosaccharides. Binding of radiolabeled, rhASM to the ASMKO macrophages was enhanced as compared with normal macrophages, consistent with their larger size and increased surface area. However, internalization of the enzyme by the ASMKO cells was markedly reduced when compared with normal cells. Studies using receptor-specific ligands to inhibit enzyme uptake revealed that in normal cells rhASM was taken up by a combination of mannose and mannose 6-phosphate receptors (MR and M6PR, respectively), whereas in the ASMKO cells the M6PR had a minimal role in rhASM uptake. Expression of M6PR mRNA was normal in the ASMKO cells, although Western blotting revealed more receptors in these cells when compared with normal. We therefore hypothesized that lipid accumulation in ASMKO macrophages led to abnormalities in M6PR trafficking and/or degradation, resulting in reduced enzyme uptake. Consistent with this hypothesis, we also found that, when rhASM was modified to expose terminal mannose residues and target mannose receptors, the uptake of this modified enzyme form by ASMKO cells was ϳ10-fold greater when compared with the "complex" type rhASM. These findings have important implications for NPD enzyme replacement therapy, particularly in the lung.Types A and B Niemann-Pick disease (NPD) 1 are lysosomal storage disorders resulting from an inherited deficiency of acid sphingomyelinase (ASM) activity (EC 3.1.4.12) (1). ASM is responsible for hydrolyzing the lipid, sphingomyelin, to ceramide and phosphocholine, and both forms of the disease are characterized by extensive lipid storage in various cells and tissues. Patients with Type A NPD follow a rapid, neurodegenerative course that leads to death by about 3 years of age, whereas Type B NPD patients have a less severe form of the disease with little or no neurological involvement. This latter form is characterized mainly by visceral organ complications, including hepatosplenomegaly and progressive pulmonary compromise (1, 2). Intermediate forms also have been described (e.g. Ref.3), revealing that ASM deficiency can lead to a wide spectrum of disease.Enzyme replacement therapy was first suggested as an approach for the treatment of lysosomal storage disorders over 30 years ago (4), although difficulties in purifying the human enzymes limited its early evaluation (5-7). However, during the past decade the isolation of genes encoding most lysosomal enzymes and the development of expression systems that produced large quantities of recombinant proteins led to the successful treatment of sev...

Safety Study of Adeno-Associated Virus Serotype 2-Mediated Human Acid Sphingomyelinase Expression in the Nonhuman Primate Brain

“…This is concerning because partial secretion of ASM and uptake by neighboring cells has been previously documented to occur (Schissel et al, 1998). However, the efficiency of this process seems to be quite low in normal macrophages and even worse in macrophages from NPD knockout mice (Dhami and Schuchman, 2004). Because expectations of efficacy in gene therapy for LSDs in general, and NPD in particular, rest on the thesis that enzyme is secreted and subsequent taken up by neighboring cells, we expected to see significant augmentation of ASM activity in regions of brain distal to those domains demonstrated to contain vector DNA.…”

Novel first-dose adverse drug reactions during a phase I trial of olipudase alfa (recombinant human acid sphingomyelinase) in adults with Niemann–Pick disease type B (acid sphingomyelinase deficiency)

“…The slow clearance of olipudase alfa from the circulation may be a result of its binding to lipoprotein particles or cell membranes 20 and/or impaired targetcell uptake via mannose-6-phosphate receptors. 21 Interestingly, the types of ADRs observed upon first exposure to olipudase alfa have not been reported in patients with Gaucher disease receiving imiglucerase ERT, even though ceramide is a catabolite of glucosylceramide, the enzyme substrate. The rapid cellular uptake of imiglucerase from plasma (t 1/2 <10 min), coupled with the 100-fold lower plasma concentration of glucosylceramide relative to sphingomyelin, may limit the amount of ceramide formed in the circulation in patients with Gaucher disease.…”

Delivery of Acid Sphingomyelinase in Normal and Niemann-Pick Disease Mice Using Intercellular Adhesion Molecule-1-Targeted Polymer Nanocarriers

“…It is noteworthy that coupling recombinant ASM to anti-ICAM polymer nanocarriers (150 -300 nm in diameter) leads to efficient internalization of anti-ICAM/ASM nanocarriers by cell adhesion molecule-mediated endocytosis, a unique pathway, distinct from clathrin-and caveolar-mediated endocytosis, which fail to efficiently internalize naked ASM in disease cells (Muro et al, 2003b(Muro et al, , 2006bDhami and Schuchman, 2004) and carriers larger than 200 nm in diameter (Rejman et al, 2004). Anti-ICAM/ASM nanocarriers traffic to lysosomes in disease cells and recover sphingomyelin levels (Muro et al, 2006b), necessary for further development of this promising strategy.…”