School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia.
Department of Biochemistry & Molecular Biology Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia.
ACS Chem Biol. 2021 Jun 18;16(6):973-981. doi: 10.1021/acschembio.1c00006. Epub 2021 May 14.
Chemokines are secreted proteins that regulate leukocyte migration during inflammatory responses by signaling through chemokine receptors. Full length CC chemokine ligand 14, CCL14(1-74), is a weak agonist for the chemokine receptor CCR1, but its activity is substantially enhanced upon proteolytic cleavage to CCL14(9-74). CCL14 is O-glycosylated at Ser7, adjacent to the site of proteolytic activation. To determine whether glycosylation regulates the activity of CCL14, we used native chemical ligation to prepare four homogeneously glycosylated variants of CCL14(1-74). Each protein was assembled from three synthetic peptide fragments in "one-pot" using two sequential ligation reactions. We show that while glycosylation of CCL14(1-74) did not affect CCR1 binding affinity or potency of activation, sialylated variants of CCL14(1-74) exhibited reduced activity after treatment with plasmin compared to nonsialylated forms. These data indicate that glycosylation may influence the biological activity of CCL14 by regulating its conversion from the full-length to the truncated, activated form.
趋化因子是分泌蛋白，通过趋化因子受体信号传导来调节炎症反应期间的白细胞迁移。全长 CC 趋化因子配体 14（CCL14）（1-74）是趋化因子受体 CCR1 的弱激动剂，但在蛋白水解切割为 CCL14（9-74）后，其活性大大增强。CCL14 在 Ser7 处发生 O-糖基化，该位点紧邻蛋白水解激活位点。为了确定糖基化是否调节 CCL14 的活性，我们使用天然化学连接来制备 CCL14（1-74）的四种均匀糖基化变体。每种蛋白质都是使用两个连续的连接反应从三个合成肽片段在“一锅”中组装而成的。我们表明，尽管 CCL14（1-74）的糖基化不影响 CCR1 结合亲和力或激活效力，但与非唾液酸化形式相比，在用纤溶酶处理后，CCL14（1-74）的唾液酸化变体的活性降低。这些数据表明，糖基化可能通过调节全长到截短的激活形式的转化来影响 CCL14 的生物学活性。
