奈米探針結合親和純化質譜技術應用於第八型半乳糖凝集素結合的醣蛋白解析

Abstract

半乳糖凝集素是一種會結合β-半乳醣的凝集素，透過其蛋白質與醣辨認及蛋白質間的交互作用，在免疫反應及癌症過程中扮演了重要的角色。由於半乳糖凝集素與其結合的蛋白質之間的交互作用力相當微弱，在特定的生理及病理的條件下，直接鑑定出內生性的結合蛋白質及辨認的醣型結構相當不容易。有著兩個不同認醣區域的第八型半乳糖凝集素 (Galectin-8, Gal-8)，目前只有被報導過會抑制細胞黏附、影響細胞移動及造成細胞程式凋亡作用。兩個認醣區域對於不同寡糖的解離常數已經被測量得到。然而，不像已被透徹研究的第一型及第三型半乳糖凝集素，詳細的調控機制、直接辨認的醣型以及重要的調控信息傳遞仍是未知。 在此我們發展了一種策略，利基於奈米碳針的親和純化結合質譜分析以研究第八型半乳糖凝集素於大腸直腸癌中的交互作用網及醣型的辨認。製備出鍵結著全長、N端認醣區域、及C端認醣區域第八型半乳糖凝集素的磁性奈米碳針，用以純化他們的交互作用蛋白質。透過CRAPome資料庫、控制組奈米碳針及膜蛋白與細胞間質的篩選，屏除非專一性吸附的蛋白質可得到真正交互作用的蛋白質。總計289個蛋白質被鑑定為可信的交互作用分子，包含許多文獻同樣報導過的交互作用蛋白質，例如與細胞黏附及細胞間交互作用有關的整合素族群、CD166及CD44。第八型半乳糖凝集素可能辨認的醣胜肽可以透過胜肽層級的交互作用實驗獲得，並配合切醣酵素水解或完整的醣基結構鑑定。總計585條醣胜肽 (157種蛋白質) 被鑑定，其中有509條醣胜肽含有N-乙醯乳糖胺或唾液酸化N-乙醯乳糖胺。與蛋白質層級的實驗結果交集後，共273條醣胜肽 (36種蛋白質) 被鑑定，其中包含一個與腫瘤生成相關的酪氨酸受體—表皮生長因子受體，被發現與第八型半乳糖凝集素交互作用的N-醣基團，是一個在N568位置上帶有核心岩藻糖基化及唾液酸化的混合醣型。在第八型半乳糖凝集素刺激下，表皮生長因子受體在與受體二聚化及泛素化作用相關的位置—T693及Y1068皆有磷酸化表現暫時性改變的現象，而其信息下游的STAT3同樣也有改變。這說明了第八型半乳糖凝集素調控大腸直腸癌，可能是透過胞外的蛋白質與表皮生長因子受體的醣類交互作用，再刺激自我磷酸化以及下游信息傳遞。我們展示了基於奈米碳針的親和純化結合質譜分析的策略可以有效的鑑定交互作用分子以及醣基結構，這些將可以提供我們許多資訊，以了解胞外的半乳糖凝集素的醣類辨識作用。

Galectins, β-galactosides-binding lectins, play important roles in immune response and cancer biology. Due to the weak interaction between galectins and carbohydrates on recognized glycoproteins, it is challenging to directly identify endogenous binding partners and the recognition glycotope under physiological and pathological conditions. Galectin-8 (Gal-8) with two tandem-repeat types of carbohydrate-recognition domains (CRD) has been reported for inhibition of cell adhesion, influence migration, and induced apoptosis. The binding oligosaccharides and the dissociation constants with N- and C-terminal CRDs of Gal-8 have been determined. Unlike the well-studied galectin-1 (Gal-1) and galectin-3 (Gal-3), however, the direct binding partners and recognition glycotopes critical to Gal-8 signaling are still unclear. In this study, we developed a nanoprobe-based affinity purification strategy combined with mass spectrometry analysis to study the interactome and glycotope recognition of Gal-8 in colorectal cancer cells. Full length, N- and C-terminal Gal-8@MNP nanoprobes as well as BSA@MNP were fabricated to purify the Gal-8 binding proteins. True binding proteins were determined by filtering out non-specific binding proteins from proteins extracted from BSA@MNP and CRAPome database as well as annotation of plasma membrane protein and extracellular matrix by GO database. Total of 289 proteins were identified as confident Gal-8 interacting partners, including identification of several reported Gal-8 binding partners, such as integrin family, CD166 and CD44, which are known to relate to cell adhesion and cell-cell interaction. The potential glycopeptides recognized by Gal-8 was analyzed by peptide level interaction assay, following by de-glycosyalted or intact glycotope characterization. A total of 585 intact glycopeptides corresponding to 157 glycoproteins were found, among them 509 glycopeptides contained LacNAc/sialo-LacNAc. Correlated and overlaid with protein level results, total of 36 glycoproteins (273 intact glycopeptides) were identified, including the well-known tumorigenesis-related receptor tyrosine kinase, epidermal growth factor receptor (EGFR), with hybrid-type glycan with core-fucosylation and sialylation on N568. Further experiment upon recombinant Gal-8 stimulation induced temporal alteration of the phosphorylation on T693 and Y1068 of EGFR, which sites have been reported to relate to receptor dimerization and ubiquitination, and its downstream substrates STAT3. This suggested that Gal-8 may regulate CRC tumorigeneses through extracellular interaction with EGFR can further induce autophosphorylation and its intracellular signaling transduction. We demonstrated that nanoprobe-based affinity purification mass spectrometry strategy is useful to identify interacting partners and glycotopes to provide information to understand the extracellular galectin recognition.