The data from these assays were complemented by a glycan array analysis which determined that AAV1 efficiently binds to NeuAcα2-3GalNAcβ1-4GlcNAc as well as glycoproteins containing NeuAcα2-3/α2-6Galβ1-4GlcNAc N-linked glycans. Sialic acids are typically found as terminal monosaccharides of glycans carried by cell surface glycoproteins. Mucin competition was carried out by pretreating rAAV with bovine submandibular gland mucin (Sigma) for 30 min at 20°C. AAV vectors, in the presence or absence of mucin, were added to Cos-7 cells in equal volumes of DMEM for 1 h at 37°C. Cells were rinsed twice with DMEM and incubated at 37°C. Two thousand particles/cell were pretreated with mucin (concentrations up to 0.5 mg/ml), and cells were assayed for luciferase expression 24 h posttransduction. Further analysis using lectin staining and lectin competition assays identified that AAV1 and AAV6 use either α2,3-linked or α2,6-linked sialic acid when transducing numerous cell types (HepG2, Pro-5, and Cos-7). Cos-7 cells were treated with the indicated doses of N-benzyl GalNAc (A) or tunicamycin (B) for 24 h prior to transduction.
In the present study, we showed that AAV1 competes with AAV6 transduction and vice versa in cultured cells, suggesting that AAV1 and AAV6 might use the same receptor or that they share some common receptors for transduction. Evidence from different groups suggests that the state of sialylation of DCs may influence their response.17,18 We have previously studied the surface sialylation of human moDCs and we observed a significantly increased expression of sialylated structures during the differentiation of monocytes into moDCs, most probably as the result of the activity of ST3Gal.I and ST6Gal. Should you loved this post and you would want to receive more details regarding sialic acid powder suppliers (just click the next website) kindly visit our own webpage. I sialyltransferases.19 In addition, we have also observed that the removal of the sialylated structures by neuraminidase treatment diminished the moDC capacity for endocytosis,19 suggesting a triggering of DC maturation. Statistical comparison between two different groups was performed using Student’s t-test (GraphPad, GraphPad Software, La Jolla, CA). When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
We used cell-based assays to show that α2,3 and α2,6 sialic acids that are present on N-linked glycoproteins facilitate cellular transduction by both AAV1 and AAV6 vectors. The above results (Fig. (Fig.2)2) show that AAV1 and AAV6 transduce Pro-5, Cos-7, and HepG2 cells efficiently; therefore, we tested the sialic acid linkages on the surfaces of these cell lines. Similarly, SNA blocked AAV1 and AAV6 transduction on HepG2 cells and, to a lesser extent, on Cos-7 cells but not on Pro-5 cells. In addition, there was a 60-fold difference between Pro-5 cells and Lec-2 cells for AAV6 transduction while only an 8-fold difference for AAV1 transduction. While AAV6 seems to be a naturally evolved recombinant between AAV1 and AAV2 (34, 46), differing in only six amino acids in the capsid region from AAV1, both AAV1 and AAV6 vectors transduced muscle very efficiently (2, 5). However, when these vectors were tested on other tissues such as liver, AAV6 showed much higher transduction efficiency than AAV1 (16). Whether use by these viruses of identical primary receptors and different secondary receptors explains the tissue preference described above remains unknown.
For instance, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. RPMI-1640 supplemented with 10% fetal calf serum from Sigma (St Louis, MO, USA), 2 mm l-glutamine, 1% non-essential amino acids, 1% pyruvate, 100 μg/ml penicillin/streptomycin and 50 μm 2-mercaptoetanol, all from Gibco-Invitrogen (Paisley, UK) was used throughout this study for cell culture. 05 mg/ml mitomycin C for 30 min and washed three times with 10 ml RPMI-1640 medium. The viruses were removed, and the cells were washed three times with medium. All experiments were performed with triplicates for each sample and independently repeated three times. Electrophoresis was performed on ice for 45 min at 120 V in 1 × Tris-Glycine buffer. Afterwards, the Tris-Glycine gel was recovered and placed in deionized water to wash out bromophenol blue band. VRM Live – 09/24/10: Vaccine Resistance Movement Founder Joel Lord & activist/radio host Jesse Calhoun lay it all out tonite. Heparan sulfate proteoglycan (HSPG) has been suggested as the primary receptor of AAV2 (42). The HSPG interaction domain on AAV2 capsids has been identified as a clustering of basic residues, particularly R585 and R588, contributed by the icosahedral threefold-symmetry-related VP3 monomers, in the valley between the three protrusions surrounding the icosahedral threefold axis (21, 28). The attachment factor for AAV4 and AAV5 was determined to be sialic acid with different linkage forms (2,3 O linked versus 2,3 N linked) (19, 44), although the sialic acid capsid interaction domains have not been identified (29, 43). Besides these serotypes, identification of primary receptors for other serotypes has been lacking.
