Alzheimer's beta-secretase, beta-site amyloid precursor protein-cleaving enzyme, is responsible for cleavage secretion of a Golgi-resident sialyltransferase

Abstract

The deposition of amyloid beta-peptide (A beta) in the brain is closely associated with the development of Alzheimer's disease. A beta is generated from the amyloid precursor protein (APP) by sequential action of beta-secretase (BACE1) and gamma-secretase. Although BACE1 is distributed among various other tissues, its physiological substrates other than APP have yet to be identified. ST6Gal I is a sialyltransferase that produces a sialyl alpha 2,6galactose residue, and the enzyme is secreted out of the cell after proteolytic cleavage. We report here that BACE1 is involved in the proteolytic cleavage of ST6Gal I, on the basis of the following observations. ST6Gal I was colocalized with BACE1 in the Golgi apparatus by immunofluorescence microscopy, suggesting that BACE1 acts on ST6Gal I within the same intracellular compartment. When BACE1 was overexpressed with ST6Gal I in COS cells, the secretion of ST6Gal I markedly increased. When APP(SW) (Swedish familial Alzheimer's disease mutation), a preferable substrate for BACE1, was coexpressed with ST6Gal I in COS cells, the secretion of ST6Gal I significantly decreased, suggesting that that the beta-cleavage of overexpressed APP(SW) 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 in vitro. Thus, we conclude that BACE1 is responsible for the cleavage and secretion of ST6Gal I.

Figure 1

Cleavage of ST6Gal I for secretion is sequence specific. (A) Schematic structure of rat ST6Gal I and its soluble secreted form. (B) Site-directed mutagenesis was carried out with primers incorporating the desired mutations with the QuickChange system (Stratagene). COS cells transiently expressing wild-type or mutant ST6Gal I in the pSVL expression vector, were pulse labeled and chased for 6 h for further immunoprecipitation analysis by using anti-ST6Gal I antibody (C, cell lysates; M, media.). (C) The percentage of secretion (mean ± SD, n = 3), was defined as the ratio of the radioactivity of soluble ST6Gal I in the medium to the total ST6Gal I radioactivity (medium + cells). Significant difference is indicated with an asterisk (P < 0.05).

Figure 2

Intracellular colocalization of ST6Gal I and BACE1-myc in HEK293 cells. HEK293 cells that stably expressed ST6Gal I were transfected with cDNA of BACE1-myc. Cells were stained with anti-myc and anti-ST6Gal I antibodies and then subjected to confocal fluorescence microscopy. (Top) Anti-myc immunostaining (red). (Middle) Anti-ST6Gal I immunostaining (green). (Bottom) Overlay of myc and ST6Gal I immunostaining. Their colocalization staining appears yellow. (Bar = 20 μm.)

Figure 3

Involvement of BACE1 in ST6Gal I secretion. (A) COS cells expressing rat ST6Gal I in the pSVL vector and either human BACE1 or vector were pulse labeled and chased for 3 h for further immunoprecipitation analysis by using anti-ST6Gal I antibody (C, cell lysates; M, media.) The percentage of secretion (mean ± SD, n = 3) was defined as the ratio of the radioactivity of soluble ST6Gal I in the medium to the total ST6Gal I radioactivity (medium + cells). (B) COS cells were transiently cotransfected with ST6Gal I FLAG-pSVL and either human BACE1 or vector. After 24 h of expression, soluble ST6Gal I FLAG in the media were pulled down with M2-agarose and then detected with an E41 antibody. (C) Cells expressing ST6Gal I plus either APP_SW, C99 or control pcDNA3.1 vector, were pulse labeled and chased for 6 h, and analyzed as shown in A. The percentage of secretion, taken as an average of two independent experiments, was defined as the ratio of the radioactivity of soluble ST6Gal I in the medium to the total ST6Gal I radioactivity (medium + cells). APP and APPsβ were detected with 6E10 and βNL antibodies, respectively. (D) Microsome fractions were prepared from HEK293 cells that stably express BACE1 or from their parent cells, and equivalent amounts of proteins were used for staining with SNA lectin that recognizes Siaα2,6Gal oligosaccharide structure. (For SNA lectin staining, 40 μg of protein was used for lanes 1 and 2, 10 μg for lanes 3 and 4, and 20 μg for Coomassie staining.)

Figure 4

Purified BACE1-Fc cleaves protein A-ST6Gal I in vitro. (A) Both BACE-Fc and protein A-ST6Gal I proteins, respectively, were purified from culture media of COS cells expressing those proteins by absorbing them to protein A-Sepharose and IgG-Sepharose. Reaction mixture, containing BACE-Fc and protein A-ST6Gal I and protease inhibitors for possible contaminating proteases that associate with Sepharose beads, was incubated at 37°C in the presence or absence of 100 nM BACE inhibitor (I) for 0 or 30 min with rotation. Reaction was quenched and analyzed by immunoblotting with anti-ST6Gal I antibody. The experiment was repeated four times, and the representative result is shown in A. (B) Silver-stained gel of purified BACE-Fc.