Site-Specific Chemoenzymatic Conjugation of High-Affinity M6P Glycan Ligands to Antibodies for Targeted Protein Degradation.
Site-Specific Chemoenzymatic Conjugation of High-Affinity M6P Glycan Ligands to Antibodies for Targeted Protein Degradation.
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DOI：
10.1021/acschembio.1c00751
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发表时间：
2022-11-18
影响因子：
4
通讯作者：
Wang, Lai-Xi
中科院分区：
文献类型：
--
作者：
Zhang, Xiao;Liu, Huiying;He, Jia;Ou, Chong;Donahue, Thomas C.;Muthana, Musleh M.;Su, Lishan;Wang, Lai-Xi
溶酶体靶向嵌合体（LYTACs）提供了降解感兴趣的细胞外蛋白和膜相关蛋白的机会。在这里，我们报告了一种有效的化学酶方法，该方法可以将高亲和力甘露糖-6-磷酸（M6P）聚糖配体单步和位点特异性偶联到抗体上，而不需要蛋白质工程和传统的点击反应，这些反应会引入“非自然”的部分，从而产生均匀的抗体-M6P聚糖偶联物，用于膜相关蛋白的靶向降解。以曲妥珠单抗和西妥昔单抗为模型抗体，我们发现野生型内糖苷酶S （Endo-S）可以高效地进行抗体去糖基化，同时将合成的m6p -甘氨酸唑啉中的m6p -甘氨酸以一罐方式转移到去糖基化的抗体上，从而得到结构明确的抗体- m6p -甘氨酸偶联物。用野生型endon - s2进行去糖基化，然后用endon - s2突变体（D184M）进行转糖基化，这两步程序也能有效地提供M6P聚糖-抗体偶联物。当Fc和Fab结构域都被糖基化时，化学酶方法对Fc聚糖重构具有高度特异性，西妥昔单抗的选择性Fc聚糖重构就是一个例子。SPR结合分析表明，M6P偶联物对阳离子非依赖性甘露糖-6-磷酸受体（CI-MPR）具有纳摩尔范围的结合亲和力。初步的细胞实验表明，m6p -曲妥珠单抗和m6p -西妥昔单抗结合物能够选择性地降解膜相关的HER2和EGFR。这种模块化的聚糖重塑策略有望在基于抗体的溶酶体靶向降解细胞外蛋白和膜蛋白方面找到广泛的应用。
Lysosome-targeting chimeras (LYTACs) offer an opportunity for the degradation of extracellular and membrane-associated proteins of interest. Here, we report an efficient chemoenzymatic method that enables a single-step and site-specific conjugation of high-affinity mannose-6-phosphate (M6P) glycan ligands to antibodies without the need of protein engineering and conventional click reactions that would introduce “unnatural” moieties, yielding homogeneous antibody-M6P glycan conjugates for targeted degradation of membrane-associated proteins. Using trastuzumab and cetuximab as model antibodies, we showed that the wild-type endoglycosidase S (Endo-S) could efficiently perform the antibody deglycosylation and simultaneous transfer of an M6P–glycan from a synthetic M6P–glycan oxazoline to the deglycosylated antibody in a one-pot manner, giving structurally well-defined antibody–M6P glycan conjugates. A two-step procedure, using wild-type Endo-S2 for deglycosylation followed by transglycosylation with an Endo-S2 mutant (D184M), was also efficient to provide M6P glycan–antibody conjugates. The chemoenzymatic approach was highly specific for Fc glycan remodeling when both Fc and Fab domains were glycosylated, as exemplified by the selective Fc-glycan remodeling of cetuximab. SPR binding analysis indicated that the M6P conjugates possessed a nanomolar range of binding affinities for the cation-independent mannose-6-phosphate receptor (CI-MPR). Preliminary cell-based assays showed that the M6P–trastuzumab and M6P–cetuximab conjugates were able to selectively degrade the membrane-associated HER2 and EGFR, respectively. This modular glycan-remodeling strategy is expected to find wide applications for antibody-based lysosome-targeted degradation of extracellular and membrane proteins.
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DOI：
10.1016/j.bbrc.2014.10.006
发表时间：
2014-10-31
影响因子：
3.1
作者：
Montrose, Kristopher;Krissansen, Geoffrey W.
通讯作者：
Krissansen, Geoffrey W.
影响因子：
2.9
作者：
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通讯作者：
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影响因子：
14.8
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Bondeson DP;Mares A;Smith IE;Ko E;Campos S;Miah AH;Mulholland KE;Routly N;Buckley DL;Gustafson JL;Zinn N;Grandi P;Shimamura S;Bergamini G;Faelth-Savitski M;Bantscheff M;Cox C;Gordon DA;Willard RR;Flanagan JJ;Casillas LN;Votta BJ;den Besten W;Famm K;Kruidenier L;Carter PS;Harling JD;Churcher I;Crews CM
通讯作者：
Crews CM
影响因子：
64.8
作者：
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通讯作者：
Bertozzi CR
DOI：
10.1073/pnas.1702173114
发表时间：
2017-03-28
影响因子：
11.1
作者：
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通讯作者：
Wang, Lai-Xi