Use of a monoclonal antibody to distinguish between precursor and mature forms of human lysosomal α‐glucosidase

Abstract

The maturation of lysosomal a-glucosidase in cultured human skin fibroblasts was studied using a monoclonal antibody that distinguishes between the precursor and mature forms of the enzyme.1. Monoclonal antibodies against a-glucosidase isolated from placenta were produced by the hybridoma technique [Hilkens et al. (1981) Biochim. Biophys. Acta 678, 7-11]. One of these monoclonal antibodies, that synthesized by clone 4368, reacts with the mature forms, but not with the precursor form of a-glucosidase isolated from urine.2. By means of pulse-labelling studies, it could be shown that monoclonal antibody 4368 does not react with either the intracellular or the secreted precursor of a-glucosidase from cultured human skin fibroblasts. However, the antibody does react with the intermediate and mature forms of a-glucosidase.3. Endocytosis of the precursor of a-glucosidase from urine by fibroblasts is followed by its conversion to a form with lower molecular mass. 4. After endocytosis urinary precursor a-glucosidase is converted to a form that binds to monoclonal antibody 4368. The t'Iz for this conversion is 2 h. The conversion is inhibited by addition of leupeptin to the culture medium. 5.It is concluded that a thiol proteinase is involved in the maturation of a-glucosidase in fibroblasts and the appearance of the antigenic determinant for 4368.Lysosomal a-glucosidase, like all other lysosomal enzymes of which the 'life-cycle' has been studied [l], is synthesized as a large precursor that is processed to mature forms of lower molecular mass [2]. Although very little is known about this proteolytic process, it is thought to take place in the lysosomes Two factors have led us to select a-glucosidase as a convenient model for studying the proteolytic processing of lysosomal enzymes. The first is the availability of substrate amounts of a precursor form of a-glucosidase; its isolation from human urine is described in the preceding paper [4]. The second is the availability of a monoclonal antibody that enables us to distinguish between different forms of a-glucosidase.Using the hybridoma technique [5], we have produced a number of monoclonal antibodies against placental a-glucosidase [6]. In the present paper we show that one of the antibodies, that produced by clone 4368, does not react with the precursor of a-glucosidase, whereas it does react with the intermediate and mature forms of the enzyme. We describe the use of this antibody in studying the processing of the precursor form of a-glucosidase from urine after endocytosis by cultured human skin fibroblasts and, in addition, the processing of labelled endogenous precursor. PI.Abbreviations. SDS, sodium dodecyl sulphate; PAGE, polyacrylEnzyme. a-Glucosidase (EC 3.2.1.20). amide gel electrophoresis. MATERIALS A N D METHODS Partial purification of monoclonal antibodiesMonoclonal antibodies were partially purified from the culture medium of the hybridoma cell lines by 50 % (NH,),SO, precipitation at 4 "C. The precipitate was dissolved in water, dialysed against phosphate-b...

Biochemical and pharmacological characterization of different recombinant acid α-glucosidase preparations evaluated for the treatment of Pompe disease

“…The corresponding published biochemical properties of human placentalderived GAA are provided for comparison [24][25][26][27]. Each of the three recombinant GAA preparations was biochemically active towards the synthetic α-D-glucopyranoside substrate.…”

Biosynthesis and maturation of peroxisomal beta-oxidation enzymes in fibroblasts in relation to the Zellweger syndrome and infantile Refsum disease.

“…The extracts were centrifuged for 1 hr in a Spinco centrifuge at 50,000 x g, and the supernatants were used for immunoprecipitation of labeled proteins as described (33). The antibodies used were an anti-bovine liver catalase antiserum (17), an anti-rat liver acyl-CoA oxidase IgG fraction (18,34), and an anti-rat liver 3-oxoacyl-CoA thiolase IgG fraction (34).…”

“…The antibodies used were an anti-bovine liver catalase antiserum (17), an anti-rat liver acyl-CoA oxidase IgG fraction (18,34), and an anti-rat liver 3-oxoacyl-CoA thiolase IgG fraction (34). The procedures for NaDodSO4/PAGE and fluorography were as described (33). Fig.…”

Receptor-Mediated Uptake of Acid α-Glucosidase Corrects Lysosomal Glycogen Storage in Cultured Skeletal Muscle

“…After uptake by muscle cells the precursor was converted to fully active mature enzyme. This is concluded from the fact that endocytosed acid a-glucosidase can be visualized with monoclonal antibody 4368, that only recognizes mature enzyme, and not the administered precursor (30,40). In addition, the immunocytochemical labeling pattern demonstrates that all lysosomes are reached, and not a selected subset.…”

“…Immunocytochemistry was performed according to Van Dongen et al (29), using mouse monoclonal antibodies (4368) (30) raised against acid a-glucosidase and rabbit polyclonal antibodies against P-hexosaminidase (3 1). Immune complexes were visualized with goat anti-mouse IgG conjugated to a green fluorescent dye and goat anti-rabbit IgG conjugated to a red fluorescent dye, respectively.…”