No abstract

␣-L-Iduronidase is a lysosomal hydrolase that is deficient in Hurler syndrome and clinically milder variants. Recombinant human ␣-L-iduronidase, isolated from secretions of an overexpressing Chinese hamster ovary cell line, is potentially useful for replacement therapy of these disorders. Because of the importance of carbohydrate residues for endocytosis and lysosomal targeting, we examined the oligosaccharides of recombinant ␣-Liduronidase at each of its six N-glycosylation sites. Biosynthetic radiolabeling showed that three or four of the six oligosaccharides of the secreted enzyme were cleaved by endo-␤-N-acetylglucosaminidase H, with phosphate present on the sensitive oligosaccharides and L-fucose on the resistant ones. For structural analysis, tryptic and chymotryptic glycopeptides were isolated by lectin binding and reverse phase high pressure liquid chromatography; their molecular mass was determined by matrix-assisted laser desorption-time of flight mass spectrometry before and after treatment with endo-or exoglycosidases or with alkaline phosphatase. Identification of the peptides was assisted by amino-or carboxyl-terminal sequence analysis. The major oligosaccharide structures found at each site were as follows: Asn-110, complex; Asn-190, complex; Asn-336, bisphosphorylated (P 2 Man 7 GlcNAc 2 ); Asn-372, high mannose (mainly Man 9 GlcNAc 2 , some of which was monoglucosylated); Asn-415, mixed high mannose and complex; Asn-451, bisphosphorylated (P 2 Man 7 GlcNAc 2 ). The Asn-451 glycopeptide was unexpectedly resistant to digestion by N-glycanase unless first dephosphorylated, but it was sensitive to endo-␤-N-acetylglucosaminidase H and to glycopeptidase A. The heterogeneity of carbohydrate structures must represent the accessibility of the glycosylation sites as well as the processing capability of the overexpressing Chinese hamster ovary cells.

No abstract

Secreted phosphoprotein-24 kDa (Spp24) binds cytokines of the bone morphogenetic protein/transforming growth factor-β (BMP/TGFβ) superfamily and is one of the most abundant serum phosphoproteins synthesized by the liver. Little is known about how Spp24 binding affects BMP signal transduction and osteoblastic differentiation or how this labile protein is transported from the liver to remote tissues, such as bone. When Spp24 was administered to W-20-17 mesenchymal stem cells with rhBMP-2, short-term Smad1/5 phosphorylation was inhibited, intermediate-term alkaline phosphatase (ALP) induction was blunted, and long-term mineralization was unaffected. This supports the hypothesis that Spp24 proteolysis restricts the duration of its regulatory effects, but offers no insight into how Spp24 is transported intact from the liver to bone. When Spp24 was immunopurified from serum and subjected to native PAGE and Western blotting, a high molecular weight band of >500 kDa was found. Under reducing SDS-PAGE, a 24 kDa band corresponding to monomeric Spp24 was liberated, suggesting that Spp24 is bound to a complex linked by disulfide bonds. However, such a complex cannot be disrupted by 60 mM EDTA under non-reducing condition or in purification buffers containing 600 mM NaCl and 0.1% Tween-20 at pH 2.7-8.5. LC-MS/MS analysis of affinity-purified, non-reducing SDS-PAGE separated, and trypsin digested bands showed that the Spp24 was present in a complex with three α(2) -macroglobulins (α(2) -macroglobulin [α(2) M], pregnancy zone protein [PZP] and complement C3 [C3]), as well as ceruloplasmin and the protease inhibitor anti-thrombin III (Serpin C1), which may protect Spp24 from proteolysis.