Crenomytilus grayanus貽貝凝集素探針有望用於偵測非還原端半乳糖與N-乙酰半乳糖胺數量在疾病和血球老化的改變

Abstract

從貽貝中分離出來的Crenomytilus grayanus 凝集素(CGL)在系統發生樹上具有其獨特性，除了MytiLec家族外，對大多數已鑑定的凝集素沒有保守性。通過結構分析，CGL是由穩定的β鏈結構所組成的二聚體，並且在每個單體上具有三個與糖結合的位點。CGL擁有對半乳（Gal）、N-乙醯半乳糖胺（GalNAc）和球三糖的多價結合，而被歸類為半乳糖結合凝集素家族的成員。此外，許多研究表明細胞表面非還原端半乳糖/N-乙醯半乳糖胺抗原的數量變化與疾病進展、紅血球細胞老化和血小板衰老有關。因此，這些特點激發了我們開發CGL作為探測細胞表面非還原端半乳糖/N-乙醯半乳糖胺抗原變化的探針，以利臨床診斷。 在這份報告中，我們確定了CGL對於酸鹼值和熱的穩定性。CGL在酸鹼值6-9的範圍內有介於60-70℃的熔解溫度並且在過低的酸鹼值下變得不穩定而無法測得熔解溫度。然而在CGL與半乳糖結合後，又能夠在低酸鹼值的環境下測得熔解溫度。在專一性方面，CGL在聚醣陣列上能識別大多數具有非還原端半乳糖和半乳糖第二號碳上帶有修飾的醣類，但不包含Galβ1-3GalNAc抗原（TF抗原）和大部分Lewis聚醣。CGL也未能識別具有末端帶有唾液酸、葡萄糖、N-乙醯葡萄糖胺和甘露糖的聚醣。 我們的目標是通過N-羥基琥珀醯亞胺(NHS)的化學基團，將生物素或螢光標記在CGL上，來製作監測半乳糖的探針。藉由電噴灑游離液相層析串聯二次質譜分析的結果對比CGL的結構， CGL表面上的六個賴氨酸(Lys7, Lys13, Lys29, Lys55, Lys142 和 Lys 144)可能是被標記的位點，其在空間上不干擾CGL與受質的結合。而由於海拉(HeLa)細胞表面醣類結構為已知，我們測試了CGL探針檢測其糖鏈末端唾液酸被去除時所暴露的半乳糖。通過使用流式細胞術和酵素聯凝集素分析法（ELLA）觀察到神經氨酸酶處理後的細胞與CGL的結合訊號增加。這些努力提供了具有良好特性及用於簡單操作的CGL探針和揭示精確的診斷。

Crenomytilus grayanus lectin (CGL) was isolated from mussel and was unique on the phylogenic tree with no conservation to most identified lectin except MytiLec family. From structural analysis, CGL formed a stable β strand structure as dimer and had three glycan-binding sites on each monomer. Interestingly, CGL represented substrate multivalency toward galactose (Gal), N-acetyl galactosamine (GalNAc) and globotriose, thereby being regarded as a member of galactose-binding lectin family. In addition, a number of studies demonstrated that the changes of non-reducing end Gal/GalNAc antigens on the cell surface were highly associated with disease progression, blood cell aging, and platelet senescence. Therefore, the feature of CGL inspired us to develop a probe for detecting the dynamics of cell surface Gal/GalNAc that benefit the clinical diagnosis. In this report, we determined the pH stability and thermo-stability of CGL. The Tm value of CGL was 60 – 70℃ in the pH range of 6 – 9 and CGL became unstable with undetermined Tm at lower pH environment. However, the Tm can be measured in lower pH value while CGL had bound to galactose. On substrate specificity, CGL was found to recognize most glycans with galactose and galactose derivatives with C-2 modification exposed on the non-reducing end by glycan array except Galβ1-3GalNAc antigen (TF antigen) and most of Lewis glycans. CGL also failed to recognize the glycans with terminal sialic acid, glucose, N-acetyl glucosamine and mannose. With respect to the probe, we aimed to monitor the galactose by conjugating biotin or fluorescence on CGL through NHS-based linker. ESI-LC-MS/MS results and CGL structure confirmed that labeling sites were six lysines (Lys7, Lys13, Lys29, Lys55, Lys142 and Lys 144) on the surface of CGL which did not spatially interfere with substrate binding of CGL. Owing to the known structure of HeLa cell surface glycans, we tested the CGL probe to detect galactose that was exposed while the terminal sialic acid was removed. Indeed, we observed that the signal of CGL probe dramatically increased after neuraminidase treatment by using flow cytometry and enzyme-linked lectin assay (ELLA). These efforts provide well-characterized and facile manipulated CGL based probe and shed light on a precise diagnosis.