A polyclonal antibody, E41, which specifically recognizes the N terminus of soluble ST6Gal I, was prepared, by immunizing a synthetic peptide, EFQMPKC, which was conjugated with keyhole limpet hemocyanin (21)

A polyclonal antibody, E41, which specifically recognizes the N terminus of soluble ST6Gal I, was prepared, by immunizing a synthetic peptide, EFQMPKC, which was conjugated with keyhole limpet hemocyanin (21). Expression Plasmid. ST6Gal I significantly decreased, suggesting that that this -cleavage of overexpressed APPSW competes with ST6Gal I processing. In addition, BACE1-Fc (Fc, the hinge and constant region of IgG) chimera cleaved protein A-ST6Gal I fusion protein (12, 13). Northern blot analysis showed that BACE1 mRNA is usually expressed in most peripheral tissues, including those of the brain, but its physiological substrates other than APP have yet to be recognized. Many glycosyltransferases are type II membrane proteins and are retained in the Golgi apparatus for oligosaccharide biosynthesis (14). Some of these enzymes are then cleaved by an endogenous protease, or proteases, and secreted out of the cell. Indeed, many glycosyltransferases have been found as extracellular soluble forms in bodily fluids such as serum, colostrum, and milk (15C19). It has been well documented that this proteolytic cleavage and secretion of glycosyltransferases into bodily fluids are affected by numerous pathological conditions such as malignant transformation and inflammation, but the endogenous protease responsible for the cleavage and secretion has not yet been recognized (20). In the present study, we demonstrate that BACE1 is usually involved in the proteolytic cleavage of a Golgi-resident sialyltransferase, ST6Gal I, that produces a sialyl2,6galactose residue. Materials and Methods Materials. Tissue culture media and reagents, including DMEM and Lipofectin, were purchased from Invitrogen. Protein A Sepharose Fast Circulation was purchased from Amersham Pharmacia Biotech. Columns for DNA purification were obtained from Qiagen (Chatsworth, CA.). Protein molecular weight requirements were purchased from Bio-Rad. [35S]Express protein-labeling mix was purchased from DuPont/NEN. BACE inhibitor, KTEEISEVN(Sta)VAEF, was purchased from Bachem. A polyclonal antibody, E41, which specifically recognizes the N terminus of soluble ST6Gal I, was prepared, by immunizing a synthetic peptide, EFQMPKC, which was conjugated with keyhole limpet hemocyanin (21). Expression Plasmid. For transient transfection experiment, ST6Gal I-pSVL and ST6Gal I FLAG-pSVL were constructed as explained previously (22). For a stable expression experiment, ST6Gal I FLAG-pcDNA3.1 was constructed by excision of ST6Gal I FLAG from ANA-12 ST6Gal I-pBluescript with (SNA) Lectin Blotting. Microsome fractions were prepared from HEK293 cells that stably express BACE1 or from their parent cells, and equivalent amounts of proteins (10 or 40 g for ANA-12 SNA lectin staining) were subjected to 4C20% gradient SDS/PAGE and transferred to nitrocellulose membrane. The blotted membrane was incubated with 1% ANA-12 BSA in 10 mM sodium phosphate buffer (pH 7.2), incubated with 10 g/ml of SNA-horseradish peroxidase (EY Laboratories), and then visualized by using chemiluminescent substrate. Twenty micrograms of each microsome fraction were also subjected to 4C20% gradient SDS/PAGE and then stained with Coomassie ANA-12 (Wako Biochemicals, Osaka). BACE Assay. Both BACE-Fc and protein A-ST6Gal I proteins were purified from 20 ml of culture media of COS cells that transiently expressed these proteins by Rabbit Polyclonal to OR2A5/2A14 absorbing them to 20 l of protein A-Sepharose and IgG-Sepharose (50% suspension in PBS), respectively. Reaction mixture contained 50 mM sodium-acetate buffer (pH 4.5), 1 l of BACE-Fc and protein A-ST6Gal I preparation, and protease inhibitors for possible contaminating proteases that associate with Sepharose beads [Complete (Roche)], 10 M pepstatin/1 M Leupeptin/1 mg/ml of pepstatin/2 M Amastatin] with or without 30 or 100 nM -secretase inhibitor (Bachem). The mixture was incubated at 37C for 0 or 30 min with rotation. Reaction was quenched and the product was analyzed by immunoblotting with anti-ST6Gal I antibody. Results We have studied ST6Gal I as a model protein for understanding the molecular mechanisms of the cleavage secretion of Golgi.