Structure of the lysosomal neuraminidase–β-galactosidase–carboxypeptidase multienzymic complex

Abstract

Lysosomal neuraminidase (sialidase; EC 3.2.1.18) and beta-galactosidase (EC 3.2.1.23), together with a carboxypeptidase, the so-called 'protective protein', were co-purified from the human placenta by affinity chromatography on a concanavalin A-Sepharose column followed by a thiogalactoside-agarose affinity column for beta-galactosidase. Analysis of the purified material by gel-filtration h.p.l.c. revealed three distinct molecular forms, all with high beta-galactosidase specific activity, but only the largest one expressed neuraminidase activity. Rechromatography of each individual species separately indicated that all three are in fact part of an equilibrium system (the neuraminidase-beta-galactosidase-carboxypeptidase complex or NGC-complex) and that these species undergo slow conversion into one another through dissociation and association of protomeric components. Each species was sufficiently stable for the determination of their hydrodynamic properties by gel-filtration h.p.l.c. and sedimentation velocity. The largest species had an apparent sedimentation coefficient S20.w, of 18.8 S and a Stokes' radius of 8.5 nm, giving a molecular mass of 679 kDa and a fractional ratio, f/f min, of 1.47. The latter value indicates that the macromolecule is asymmetric or highly hydrated. This large species is composed of four types of polypeptide chains of molecular mass 66 kDa (neuraminidase), 63 kDa (beta-galactosidase), 32 kDa and 20 kDa (carboxypeptidase heterodimer). The 32 kDa and 20 kDa protomers are linked together by a disulphide bridge. Glycopeptidase F digestion of the NGC-complex transformed the diffuse 66-63 kDa band on the SDS gel into two close but sharp bands at 58 and 56 kDa. The two smaller species which were separated on the h.p.l.c. column correspond to tetrameric and dimeric forms of the 66-63 kDa protomers and express exclusively beta-galactosidase activity. Treatment of the NGC-complex with increasing concentrations of guanidinium hydrochloride up to 1.5 M also resulted in dissociation of the complex into the same smaller species mentioned above plus two protomers of molecular mass around 60 and 50 kDa. A model of the largest molecular species as a hexamer of the 66-63 kDa protomers associated to five carboxypeptidase heterodimers (32 kDa and 20 kDa) is proposed

Isolation and characterisation of sialidase from a strain of Streptococcus oralis

“…It was not possible to purify cholerae has demonstrated that this enzyme forms a multi-enzyme complex, which contains other glycosidic and proteolytic activities [36]. Sialidase complexed with other enzymes, including â-galactosidase, is also found in human lysosomes [37]. A similar association may be present with respect to the S. oralis sialidase, whereby glycosidic and proteolytic activities form a multi-enzyme complex.…”

Clinical and Molecular Heterogeneity in Hereditary β-Galactosidase Deficiency

“…This protein is incorporated into a multimolecular complex consisting of protective protein and neuraminidase [18]. No molec ular mechanism is known of interactions among these proteins.…”

Mouse model for the lysosomal disorder galactosialidosis and correction of the phenotype with overexpressing erythroid precursor cells.

“…This serine carboxypeptidase (Galjart et al 1988;Rawlings and Barrett 1994) has at least two distinct functions. First, as demonstrated by copurification experiments (Verheijen et al 1982;Yamamoto and Nishimura 1987;Potier et al 1990;Scheibe et al 1990;Hubbes et al 1992), it associates with the two glycosidases, thereby modulating their intralysosomal stability and activity (Hoogeveen et al 1983;van der Horst et al 1989;Galjart et al 1991). Second, the protein is catalytically active as a lysosomal cathepsin at acidic pH, as well as a deami- Cotresponding authot. dase/esterase at neutral pH, and has been shown to par ticipate in the inactivation of selected neuropeptides like substance P, oxytocin, and endothelin I (Jackman et al 1990;Galjart et al 1991;Jackman et al 1992;Hanna et al 1994).…”