Human neuraminidase 1 (NEU1) is a
lysosomal glycosidase that cleaves
the terminal sialic acids of sialylglycoconjugates. NEU1 is biosynthesized
in the endoplasmic reticulum (ER) lumen as an N-glycosylated
protein. NEU1 also associates with cathepsin A (CTSA) in ER, migrates
to lysosomes, and exerts catalytic activity. Extraordinary in cellulo
crystallization of NEU1 protein in ER despite carrying three N-glycans per molecule at N186, N343, and N352, respectively,
were observed when the single human NEU1 gene was overexpressed in
mammalian cells. In this study, we first purified the NEU1 from the
isolated crystals produced by the HEK293 NEU1-KO
cell transiently overexpressing the normal NEU1 and
found that the N-glycans were high-mannose or complex
types carrying terminal sialic acids. The result suggests that a part
of NEU1 crystals were formed or transported to the Golgi apparatus.
Second, we compared the effects of single amino acid substitution
at the N-sequons, including N186Q, N343Q, and N352Q,
each one N-glycan reduction from one NEU1 molecule.
We demonstrated that N186Q mutant protein with low enzyme activity
and formed a few amounts of smaller crystals. The N343Q mutant exhibited
half of the normal intracellular activity, but the numbers and sizes
of crystals were almost the same as those of normal NEU1. The N352Q
mutant exhibited almost the same activity as the normal enzyme. The
numbers of the N352Q crystals were smaller than those of normal NEU1.
According to these findings, the N186Q NEU1 protein should have lower
stability in ER due to abnormal folding. The second N-glycan at the N343-sequon has little effect on self-aggregation
of NEU1. The third N-glycan at the N352-sequon contributes
to the self-aggregation of NEU1. We also demonstrated that the three
NEU1 mutants associate with the relatively excessive CTSA and migrate
to lysosomes.
Lysosomal storage diseases (LSDs) are inborn errors caused by genetic defects of lysosomal enzymes associated with the excessive accumulation of natural substrates and neurovisceral manifestations. Until now, enzyme replacement therapy (ERT) with human lysosomal enzymes produced by genetically engineered mammalian cell lines has been applied clinically to treat several LSDs. ERT is based on the incorporation of N-glycosylated lysosomal enzymes through binding to glycan receptors on the surface of target cells and delivery to lysosomes. However, ERT has several disadvantages, including di‹culty in mass producing human enzymes, dangers of pathogen contamination, and high cost. Recently, we have succeeded in producing transgenic silkworms which overexpress human lysosomal enzymes in silk glands, and have puriˆed active and functional enzymes from middle silk glands and cocoons. Silk gland-and cocoonderived human enzymes carrying high-mannose and pauci-mannose N-glycans are endocytosed by monocytes via the mannose receptor pathway; these were then delivered to lysosomes. Human cathepsin A (Ctsa) precursor proteins puried from the cocoons have been found to suppress microglial activation in the brains of Ctsa-deˆcient mice; this deˆciency is caused by a splicing defect, and serves as a galactosialidosis model associated with the combination of a deˆciency of lysosomal neuraminidase 1 (NEU1) and the accumulation of sialyloligosaccharides. Transgenic silkworms overexpressing human lysosomal enzymes in silk glands could serve as a future bioresource to provide safe therapeutic enzymes for the treatment of LSDs. The combination of recent developments in transglycosylation technology with microbial endoglycosidases will aid in the development of therapeutic glycoproteins as bio-medicines.
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