Elimination of abnormal sialylglycoproteins in fibroblasts with sialidosis and galactosialidosis by normal gene transfer and enzyme replacement

Abstract

Sialidosis and galactosialidosis are lysosomal storage diseases caused by the genetic defects of lysosomal sialidase (neuraminidase-1; NEU1) and lysosomal protective protein/cathepsin A (PPCA), respectively, associated with a NEU1 deficiency, excessive accumulation of sialylglycoconjugates, and development of progressive neurosomatic manifestations; in addition, the latter disorder is accompanied by simultaneous deficiencies of beta-galactosidase and cathepsin A. We demonstrated that a few soluble N-glycosylated proteins carrying sialyloligosaccharides sensitive to glycopeptidase F (GPF) can be specifically detected in cultured fibroblasts from sialidosis and galactosialidosis cases by blotting with a Maackia amurensis (MAM) lectin. We also examined the therapeutic effects of normal gene transfer and enzyme replacement by evaluating the decreases in sialylglycoconjugates accumulated in fibroblasts with these NEU1 deficiencies. The specific N-glycosylated proteins detected on MAM lectin blotting as well as the granular lysosomal fluorescence due to an avidin-FITC/biotinylated MAM lectin conjugate in sialidosis and galactosialidosis fibroblasts disappeared in parallel with the restoration of the intracellular NEU1 activity after transfection of the recombinant NEU1 fused to HA tag sequence and the wild-type PPCA cDNA as well as administration of the recombinant PPCA precursor protein. The detection method for the abnormal sialylglycoproteins in cultured cells involving MAM lectin was demonstrated to be useful not only for biochemical and diagnostic analyses of NEU1 deficiencies but also for therapeutic evaluation of these conditions.

Recent progress in development of transgenic silkworms overexpressing recombinant human proteins with therapeutic potential in silk glands

“…The current authors previously reported that sialylglycoconjugates, including sialyloligosaccharides, accumulate in fibroblasts from patients with GS (28). The addition of R8-CTSA to culture medium caused a marked reduction in the sialylglycoconjugates that accumulated in fibroblasts from a patient with GS according to Maackia amurensis (MAM) lectin staining, which recognizes and binds to terminal sialic acid residues of sialylglycoconjugates (28). Thus, R8-CTSA can be incorporated into fibroblasts via macropinocytosis with an R8 tag as a CPP, it can be delivered to lysosomes, and it can activate NEU1 to degrade accumulated sialyloligosaccharides, suggesting that R8-CTSA would therapeutically benefit patients with GS.…”

“…The protection of NEU1 and GLB was also found to be partly restored. The current authors previously reported that sialylglycoconjugates, including sialyloligosaccharides, accumulate in fibroblasts from patients with GS (28). The addition of R8-CTSA to culture medium caused a marked reduction in the sialylglycoconjugates that accumulated in fibroblasts from a patient with GS according to Maackia amurensis (MAM) lectin staining, which recognizes and binds to terminal sialic acid residues of sialylglycoconjugates (28).…”

Acidic Sialidase Activity Is Aberrant in Obese and Diabetic Mice

“…however, aberrant NEU1 activity is associated with LSDs, autoimmune diseases and the malignancy and metastasis of cancer cells. 3) Furthermore, therapeutic experimental trials of NEU1 for treating LSDs 28) and studies of therapeutic approaches targeting NEU1 to treat several cancers 29) and immune diseases 10) have recently been conducted. A detailed investigation into the molecular mechanisms of NEU1 involvement in obesity might reveal novel physiological and pathological roles of NEU1 and contribute towards novel strategies for treating obesity.…”

Accumulation of free complex-type N-glycans in MKN7 and MKN45 stomach cancer cells

“…The PCR fragment was digested with EcoRI/XhoI and was cloned into pCMV-Tag4 (Stratagene) to generate the FLAG-epitope at the C-terminus in frame. The C-terminal tagging of Neu1 was previously used for functional studies of Neu1 protein [17]. For human Neu2 ORF, original pGEX4T-NEU2 plasmid [18] was provided by Dr. Soichi Wakatsuki [High Energy Accelerator Research Organization (KEK), Tsukuba, Japan].…”