by For hCMEC/D3 cells, treatment with STNA resulted in a ~1.6-fold enhancement of the crosslinking band intensity when normalized to actin (Figure 4b and 4c). Lewis X antibody and of endoglin. This protocol can be applied generally to sialic acid-mediated relationships and will facilitate recognition of sialic acid binding partners. Sialic acids are involved in the rules of a multitude of relationships.1 Despite these essential tasks, identifying sialic acid-dependent interactions remains difficult because of the transient nature. As a solution, we while others have reported use of photocrosslinking sialic acid analogs that can be used to covalently capture sialic acid-dependent relationships.2-4 These analogs are metabolically incorporated into cellular glycoconjugates where they can be used to study sialic acid-dependent relationships in a native setting. To expose sialic acid bearing the diazirine photocrosslinking group within the N-acyl part chain, we tradition cells having a related cell-permeable N-acyl-modified N-acetyl-d-mannosamine (ManNAc) analog. Previously, we showed that mammalian cells can metabolize a cell-permeable, diazirine-modified ManNAc analog, Ac4ManNDAz(2me), to diazirine-modified sialic acid, SiaDAz(2me), and add SiaDAz(2me) to glycoconjugates destined for the cell surface.3,5,6 Similarly, cells can metabolize a mannosamine with a longer linker separating the pyranose and the diazirine, Ac4ManNDAz(4me), but this process is less efficient and little SiaDAz(4me) appears on the surface of cells.6 Even though production of SiaDAz(2me) is more efficient, SiaDAz(2me) does not replace all the organic sialic acid, Neu5Ac, on the surface of mammalian cells. We have observed a range of incorporation efficiencies, from beneficial instances, where about 65 % of cell surface Neu5Ac is replaced by SiaDAz(2me), to several cell lines where cell surface SiaDAz(2me) is definitely undetectable.7 Organic Neu5Ac competes for binding to sialic acid-recognizing proteins, which may reduce the overall effectiveness of SiaDAz-mediated crosslinking. A method to selectively remove cell surface Neu5Ac while leaving SiaDAz-modified glycoconjugates undamaged would be expected to enhance production of SiaDAz-crosslinked complexes. Sialidases, also known as neuraminidases, are enzymes that remove sialic acids from glycoconjugates. Rabbit polyclonal to ZNF500 Both bacteria and viruses create extracellular sialidases that can remove sialic acids from mammalian sponsor cells,8,9 and the human being genome also encodes at least four sialidases,10 with a range of substrate specificities.11 Previous studies have shown that substitutions within the N-acyl side chain of sialic acid can affect sialidase activity neuraminidase (STNA) can remove Neu5Ac from cell surface types, while leaving SiaDAz(2me)-modified glycoconjugates undamaged. Finally, we shown the utility of this discriminating sialidase by treating cells with STNA, which enhanced SiaDAz(2me)-dependent crosslinking. RESULTS AND Conversation Chemoenzymatic synthesis of SiaDAz-labeled glycans To test sialidase specificity against SiaDAz(2me) and SiaDAz(4me) in our cell-free microwell plate assay, we 1st synthesized T-5224 SiaDAz-labeled glycans to use as sialidase substrates. We chose to perform an established one-pot chemoenzymatic reaction that has proved useful for synthesis of varied glycans with a variety of natural and unnatural sialic acids.21 In this method, the sialic acid biosynthetic precursor ManNAc or a ManNAc analog is incubated having a non-sialylated acceptor glycan and the enzymes Neu5Ac aldolase, CMP-sialic acid synthetase, and an 2-3-sialyltransferase, in order to produce the desired sialylated glycan product (Number 1). We T-5224 select biotinylated N-acetyl-d-lactosamine (LacNAc-biotin) as our acceptor glycan, and used ManNAc or a diazirine-containing analog, ManNDAz(2me) or ManNDAz(4me), to produce LacNAc-biotin revised with 2-3-linked Neu5Ac, SiaDAz(2me), or SiaDAz(4me). (Although SiaDAz(4me) is not efficiently integrated into cell surface glycoconjugates,6 we thought that analyzing the SiaDAz(4me)-LacNAc-biotin substrate in our cell-free assay could provide additional information about the molecular basis of sialidase specificity.) The glycan products were separated by HPLC to identify non-sialylated, sialylated and SiaDAz-ylated glycans (Supplementary Number 1), which were isolated and characterized by mass spectrometry. Observed neuraminidase (AUNA),24 and a LT2 neuraminidase (STNA).25 In addition, we examined three human sialidases: NEU2,26 NEU327 and NEU4.28 We confirmed that PAL could label SiaDAz-containing glycans, as upon conjugation to aminooxy-Alexa Fluor 488, SiaDAz(2me)-LacNAc-biotin and SiaDAz(4me)-LacNAc-biotin yielded higher fluorescence ideals than unsialylated LacNAc-biotin (Supplementary Number 5). Next, we used our cell-free microwell plate assay to measure the activity of sialidases toward 2-3-linked Neu5Ac. As expected,12,23,29,30 we found that all five bacterial enzymes tested were active against 2-3-linked Neu5Ac (Number 2a). Two of the mammalian sialidases, NEU2 and NEU4, showed good to moderate activity against Neu5Ac, respectively, but NEU3 showed no activity against Neu5Ac with this cell-free assay (Number 2a). T-5224 The lack of detectable activity for NEU3 is definitely consistent with its reported preference for ganglioside-like substrates.14,31 We then examined the ability of the panel of sialidases to remove diazirine-containing sialic acid analogs, SiaDAz(2me) (Number 2b) and SiaDAz(4me) (Number 2c). Two of the sialidases from pneumococcus, NanB and NanC, were able to cleave diazirine-containing sialic acids self-employed of linker size. While NEU2 was also able to cleave SiaDAz(2me) from LacNAc-biotin, it showed little to no activity against SiaDAz(4me), suggesting the longer linker may.
