O-GalNAc糖基化因其在粘蛋白中的高丰度，也被称为粘蛋白型O-糖基化，是O-糖基化修饰中最常见的类型之一。在生物体内，粘蛋白O-糖基化修饰的启动依赖于N-乙酰氨基半乳糖转移酶（Peptide: N-acetylgalactosamine transferase, ppGalNAc-T）。ppGalNAc-T能够将糖基供体尿苷二磷酸-N-乙酰氨基半乳糖（UDP-N-acetyl-α-D-galactosamine, UDP-GalNAc）中的GalNAc转移至受体蛋白的丝氨酸（Serine, Ser）或苏氨酸（Threonine, Thr）残基上，形成Tn抗原结构，从而启动粘蛋白型O-聚糖的生物合成。由于尚未发现ppGalNAc-T的同工酶，ppGalNAc-T 的生产对于大量制备黏蛋白型O-聚糖具有重要意义。因此寻找更多具有良好催化性能的新型ppGalNAc-T并通过异源表达实现酶的体外获取，将为粘蛋白体外O-糖基化修饰提供更多可选择的工具酶，同时也为该酶在生物合成及食品领域的应用奠定了理论基础。

本文首次以普通楼燕N-乙酰氨基半乳糖转移酶为研究目标，研究采用多种表达系统进行基因克隆与蛋白表达，通过酶活性检测选择更优的表达系统，并对表达条件进行优化。采用傅立叶红外光谱检测了该酶的二级结构，运用生物信息学方法分析了氨基酸序列、同源性、与其他同功能酶进行序列比对、预测蛋白的三维构象及其与底物的相互作用模式并通过实验验证该酶的底物特异性。同时，研究了AappGalNAc-T11的酶学特性，在体外成功构建了含有多位点O-糖基化修饰的Tn抗原分子。具体研究方案如下：

1. AappGalNAc-T11的基因克隆表达、纯化与酶活性测定

本文将含目的基因的质粒导入E.coli BL21表达体系，并成功进行了蛋白表达与蛋白质复性。通过使用金门克隆组装法，设计含特殊序列的引物，对目的基因进行聚合酶链式反应（Polymerase Chain Reaction , PCR‌）扩增，并采用BsaI限制性内切酶实现了信号肽序列，目的基因序列，纯化标签序列与载体pPAP001一步组装，构建了Pichia pastoris表达质粒，并成功进行了蛋白的表达与纯化。通过将目的基因采用两种表达体系进行表达，并使用超高效液相色谱（Ultra Performance Liquid Chromatography, UPLC）测定两者表达蛋白的活性，由于大肠杆菌表达的过程中形成了包涵体蛋白，且复性后较Pichia pastoris GS115表达的蛋白活性弱，因此选择表达水平和酶活性更优越的酵母表达体系表达的蛋白进行后续实验，并随后通过比较酵母表达体系诱导温度对蛋白表达的影响，优化表达过程。

AappGalNAc-T11底物特异性研究

通过对氨基酸序列分析，AappGalNAc-T11的分子式为C₂₀₂₅H₃₁₅₂N₅₈₄O₅₉₅S₂₀，理论等电点为6.51，为亲水性蛋白质；同源性分析显示该酶与锈边短嘴霸鹟（Myiozetetes Cayanensis）来源的酶具有较强的同源性，为98%，位于发育树的同一支中；同源序列比对结果显示，该酶含有保守的色氨酸（Tryptopha, Trp），先前研究表明该氨基酸的旋转异构体的改变会引起相邻活性位点的活性的改变；通过傅立叶红外光谱检测了蛋白的二级结构，其中β-折叠占13.89%，无规则卷曲占23.59%，α-螺旋占24.74%，β-转角占据了37.79%；使用软件对蛋白质的三维结构进行了预测，并以UDP-GalNAc、尿苷二磷酸半乳糖（Uridine diphosphate galactose, UDP-Gal）、尿苷二磷酸葡萄糖（Uridine diphosphate Glucose, UDP-Glc）、尿苷二磷酸-N-乙酰葡糖胺（Uridine Diphosphate N-Acetylglucosamine, UDP-GlcNAc）作为糖基供体进行分子对接，分析该酶的关键氨基酸，利用分子对接与酶促反应实验分析AappGalNAc-T11的底物特异性，结果表明AappGalNAc-T11不能转移GlcNAc、Glc、Gal至EA2上，只可转移GalNAc至底物上，说明AappGalNAc-T11具有底物特异性。

AappGalNAc-T11的酶学特性分析及其在Tn抗原合成中的应用

检测该酶的酶学特性结果表明：最适反应温度为37°C，在20°C-40°C孵育75 min后，均能保持95%以上活力，具有较强的温度稳定性；最适反应pH为9.0，在pH=3、9、11条件下，孵育5 d残余酶活力菌保留在50%以上，该酶在弱酸性和弱碱性条件下均有较好的pH稳定性；Mn²⁺对其催化EA2作为糖基受体、UDP-GalNAc作为糖基供体的反应具有显著的促进作用；在最适反应条件下测定其动力学参数，AappGalNAc-T11的K_m为1.152 mmol/L，V_max为0.02574 μmol·L^-1·min^-1，K_cat为21.145 min^-1m，K_cat/K_m为18.6 µmol^-1·min^-1，与Aerodramus fuciphagus，Nomia melanderi 、Homo sapiens来源的ppGalNAc-T2相比，V_max是其2.01倍、1.56倍、1.75倍，K_cat/K_m的值分别为其1.72倍、1.98倍、1.68倍，说明AappGalNAc-T11具有较强的亲和力和催化效率。采用改造后的SOMO-MUC5AC作为糖基受体，AappGalNAc-T11可转移最多三个GalNAc基团至SOMO-MUC5AC的O-GalNAc糖基化位点，形成Tn抗原，发掘了体外合成短链Tn抗原的工具酶。

O-GalNAc glycosylation, also termed mucin-type O-glycosylation due to its high abundance in mucins, represents one of the most prevalent forms of O-glycosylation modifications. In biological systems, the initiation of mucin O-glycosylation relies on ppGalNAc-T (polypeptide N-acetylgalactosaminyltransferase). This enzyme catalyzes the transfer of GalNAc (N-acetylgalactosamine) from the sugar donor UDP-GalNAc (uridine diphosphate-N-acetylgalactosamine) to serine (Ser) or threonine (Thr) residues on acceptor proteins, thereby forming the Tn antigen structure and initiating the biosynthesis of mucin-type O-glycans. As no isozymes of ppGalNAc-T have been identified to date, the production of ppGalNAc-T holds significant importance for large-scale preparation of mucin-type O-glycans. Consequently, the discovery of novel ppGalNAc-T with robust catalytic properties and their acquisition via heterologous expression will expand the repertoire of tool enzymes for in vitro mucin O-glycosylation, while also establishing a theoretical foundation for their applications in biosynthesis and the food industry.

This study marks the first investigation targeting the N-acetylgalactosaminyltransfer

-ase from the Apus apus. We employed multiple expression systems for gene cloning and protein production, selecting optimal systems through enzymatic activity assays followed by expression condition optimization. The secondary structure of the enzyme was characterized using Fourier transform infrared spectroscopy. Bioinformatics analyses were conducted to assess amino acid sequence features, homology, sequence alignment with functionally analogous enzymes, three-dimensional structural predictions, and substrate interaction patterns. Experimental validation was performed to determine substrate specificity. Concurrently, we investigated the enzymatic properties of AappGalNAc-T11 and successfully constructed Tn antigen molecules containing multi-site O-glycosylation modifications in vitro. The detailed research framework is outlined below:

1. Gene cloning, expression, purification, and enzyme activity assay of AappGalNAc-T11

The target gene was expressed in E.coli BL21, followed by successful protein refolding. Using the Golden Gate assembly method, primers containing specific sequences were designed for PCR amplification. The signal peptide, target gene, and purification tag sequences were ligated to the plasmid pPAP001 via BsaI restriction enzyme digestion, enabling one-step construction of a Pichia pastoris expression plasmid. Protein expression and purification were achieved in both systems. Ultra-performance liquid chromatography revealed that the E.coli-expressed protein formed inclusion bodies, and its refolded activity was inferior to that of the Pichia pastoris GS115-expressed protein. Therefore, the proteins expressed in the yeast expression system with better activity were selected for further experiments, and then the expression process was optimized by comparing the effects of the temperature of the yeast expression system on the protein expression.

Investigation of substrate specificity of AappGalNAc-T11

Amino acid sequence analysis indicated that AappGalNAc-T11 has a molecular formula of C₂₀₂₅H₃₁₅₂N₅₈₄O₅₉₅S₂₀, a theoretical isoelectric point of 6.51, and hydrophilic properties. Homology analysis showed the highest similarity (98%) with the enzyme from Myiozetetes cayanensis, clustering on the same phylogenetic branch. Sequence alignment revealed a conserved tryptophan residue, previously implicated in modulating adjacent active site conformations via rotameric shifts. FTIR analysis indicated a secondary structure composition of 13.89% β-sheets, 23.59% random coils, 24.74% α-helices, and 37.79% β-turns. The three-dimensional structure of protein was predicted by using software, and molecular docking was performed with UDP-GalNAc, UDP-Gal, UDP-Glc, UDP-GlcNAc as glycosyl donors, EA2 as glycosyl acceptor, and the key amino acids were analyzed. The specificity of AappGalNAc-T11 was analyzed by using molecular docking and enzyme-catalyzed reaction experiment. The results showed that AappGalNA-T11 could not transfer GlcNAc, Glc, Gal to EA2, but only GalNAc to the substrate, which indicated that AappGalNA-T11 had substrate specificity. 

Enzymatic characterization of AappGalNAc-T11 and its application in Tn antigen synthesis

Key enzymatic properties: Optimal temperature: 37°C; retains >95% activity after 75 min incubation at 20-40°C, indicating remarkable thermostability. Optimal pH: 9.0; retains >50% residual activity after 5 d at pH 3-11, demonstrating stability under weakly acidic/alkaline conditions. Mn²⁺ significantly enhances catalytic efficiency when using EA2 and UDP-GalNAc as substrates. Kinetic parameters (optimal conditions): K_m=1.152 mmol/L, V_max=0.02574 μmol·L⁻¹·min⁻¹, K_cat=21.145 min⁻¹, and K_cat/K_m=8.6 μmol⁻¹·min⁻¹. Compared to ppGalNAc-T2 from Aerodramus fuciphagus, Nomia melanderi, and Homo sapiens, AappGalNAc-T11 exhibits 2.01-, 1.56-, and 1.75-fold higher V_max and 1.72-, 1.98-, and 1.68-fold higher K_cat/K_m, respectively, indicating superior affinity and catalytic efficiency.

[1] 曹翠．多肽：N-乙酰氨基半乳糖转移酶的活性检测及其原核表达载体的改造与应用［D］.南京农业大学，2017.

[2] 晁然，原永波，赵惠民．构建合成生物学制造厂［J］.中国科学：生命科学，2015，45(10)：976-984．

[3] 邓会群，王惠利，杨红，等．天然产物的C-糖基化研究进展［J］．生物技术通报，2009，(05)：27-30．

[4] 高珂星，廖春华，李昇泽，等．粘蛋白1调控肿瘤细胞恶性特征的功能位点分析［J］．上海交通大学学报(医学版)，2024，44(11)：1370-1382．

[5] 李海洋，张淼，陆峰，等．真核翻译起始因子4γ2调控N-乙酰氨基半乳糖转移酶1 mRNA对胃癌细胞恶性表型的影响［J］．实用临床医药杂志，2023，27（2）：40-49．

[6] 刘宏波，王庆，安志鹏，等．MUC5AC在重症肺炎痰热壅肺证患者气道中的表达及临床意义［J］．中医药通报，2024，23（10）：40-45．

[7] 刘晓丽，龚振兴，马雪婷，等．柔嫩艾美耳球虫多肽：N-乙酰氨基半乳糖转移酶2的基因表达及转录动态分析［J］．畜牧兽医学报，2017，48(11)：2173-2180．

[8] 冉冉．金丝燕（Aerodramus mearnsi源）燕窝粘蛋白的表达及O-GalNAc糖基化修饰［D］．南京：南京农业大学，2022．

[9] 苏丽英，王慧娟，张雷，等．粘蛋白MUC-1在人胚胎胃黏膜发育中的阶段表达［J］．解剖学报，2004，(04)：446-448．

[10] 薛庆中．DNA和蛋白质序列数据分析工具(第三版)［J］．新疆农业科学，2012，49(07)：1300．

[11] 杨玲焰，刘珺，高媛，等．人多肽N-乙酰氨基半乳糖转移酶-2对胶质瘤细胞U251中MMP-2、TIMP-2 mRNA表达及细胞迁移能力的影响［J］．苏州大学学报(医学版)，2010，30(02)：227-232+216.

[12] 张嘉欣，梁书利．人乳铁蛋白在毕赤酵母中的高效分泌表达［J/OL］．现代食品科技，2025: 1-10．

[13] 章国林，胡永洲．肿瘤相关Tn抗原的合成改进［J］．中国药学杂志，2004，(11)：68-70．

[14] 周梦倩，刘敏．多肽N-乙酰氨基半乳糖转移酶在乳腺癌中的研究进展［J］．河北医学，2020，26(12)：2106-2109．

[15] Abbas S. Protein profile analysis of tungro and dwarf virus-infected rice plants using SDS-PAGE[J]. Jurnal Biologi Tropis, 2023, 23(2): 178-186.

[16] Alcaide-Ruggiero L, Cugat R, Dominguez J M. Proteoglycans in articular cartilage and their contribution to chondral injury and repair mechanisms[J]. International journal of molecular sciences, 2023, 24(13): 10824.

[17] Argueso P. Human ocular mucins: The endowed guardians of sight［J］ Advanced drug delivery reviews, 2022, 180: 114074.

[18] Ata O, Prielhofer R, Gasser B, et al. Transcriptional engineering of the glyceraldehyde-3-phosphate dehydrogenase promoter for improved heterologous protein production in Pich-ia pastoris[J]. Biotechnology and Bioengineering, 2017, 114(10): 2319-2327.

[19] Baeri A. Towards the elaboration of an in vitro test to predict the carcinogenic nature of chemical and natural compounds used in the cosmetic industry[D]. Universite Cote d'Azur, 2021.

[20] Baghban R, Farajnia S, Ghasemi Y, et al. New developments in Pichia pastoris expression system, review and update[J]. Current pharmaceutical biotechnology, 2018, 19(6): 451-467.

[21] Bai Y, Zhang S, Dong H, et al. Advanced techniques for detecting protein misfolding and aggregation in cellular environments[J]. Chemical Reviews, 2023, 123(21): 12254-12311.

[22] Bains R K, Nasseri S A, Wardman J F, et al. Advances in the understanding and exploitation of carbohydrate-active enzymes[J]. Current Opinion in Chemical Biology, 2024, 80: 102457.

[23] Bansil R, Turner B S. Mucin structure, aggregation, physiological functions and biomedical applications[J]. Current opinion in colloid & interface science, 2006, 11(2-3): 164-170.

[24] Bechtella L, Chunsheng J, Fentker K, et al. Ion mobility-tandem mass spectrometry of mucin-type O-glycans[J]. Nature Communications, 2024, 15(1): 2611.

[25] Bonser L R, Erle D J. Airway mucus and asthma: the role of MUC5AC and MUC5B[J]. Journal of clinical medicine, 2017, 6(12): 112.

[26] Borisov R S, Matveeva M D, Zaikin V G. Reactive matrices for analytical matrix-assisted laser desorption/ionization (MALDI) mass spectrometry[J]. Critical reviews in analytical chemistry, 2023, 53(5): 1027-1043.

[27] Boye O, Nicholson L, Marstall A, et al. Silver oxide promoted synthesis of alpha O-GalNAc containing glyco-amino acids: Synthesis of Core 2 containing glyco-amino acids for solid phase synthesis of glycopeptides[J]. The Journal of Organic Chemistry, 2024, 90(1): 30-34.

[28] Bredell H, Smith J J, Gorgens J F, et al. Expression of unique chimeric human papilloma virus type 16 (HPV-16) L1‐L2 proteins in Pichia pastoris and Hansenula polymorpha[J]. Yeast, 2018, 35(9): 519-529.

[29] Camara E, Landes N, Albiol J, et al. Increased dosage of AOX1 promoter-regulated expression cassettes leads to transcription attenuation of the methanol metabolism in Pichia pastoris[J]. Scientific reports, 2017, 7(1): 44302.

[30] Chang P, Li X, Lin J, et al. scFv-oligopeptide chaperoning system-assisted on-column refolding and purification of human muscle creatine kinase from inclusion bodies[J]. Journal of Chromatography B, 2022, 1209: 123410.2

[31] Chen L, Zhang B, Chen L, et al. Hydrogen-bonded organic frameworks: design, applications, and prospects[J]. Materials Advances, 2022, 3(9): 3680-3708.

[32] Cheng F, Wang C J, Gong X X, et al. Assembly and engineering of BioBricks to develop an efficient NADH regeneration system[J]. Applied and Environmental Microbiology, 2025, 91(1): e01041-24.

[33] Cheng G, Lyu Y, Ran R, et al. Expression and in vitro glycosylation of recombinant edible bird nest (EBN)mucin[J]. Food Materials Research, 2024, 4(1).

[34] Coelho H, Rivas M, Grosso A S, et al. Atomic and specificity details of mucin 1 O-glycosylation process by multiple polypeptide GalNAc-transferase isoforms unveiled by NMR and molecular modeling[J]. Jacs Au, 2022, 2(3): 631-645.

[35] Cox K E, Liu S, Lwin T M, et al. The mucin family of proteins: candidates as potential biomarkers for colon cancer[J]. Cancers, 2023, 15(5): 1491.

[36] De Las Rivas M, Paul Daniel E J, Coelho H, et al. Structural and mechanistic insights into the catalytic-domain-mediated short-range glycosylation preferences of GalNAc-T4[J]. ACS Central Science, 2018, 4(9): 1274-1290.

[37] Deng X, Liao J, Zhao Z, et al. Distribution and speciation of selenium in soybean proteins and its effect on protein structure and functionality[J]. Food Chemistry, 2022, 370: 130982.

[38] Durin Z, Houdou M, Legrand D, et al. Metalloglycobiology: The power of metals in regulating glycosylation[J]. Biochimica et Biophysica Acta (BBA)-General Subjects, 2023, 1867(9): 130412.

[39] Dutta D, Mandal C, Mandal C. Unusual glycosylation of proteins: Beyond the universal sequon and other amino acids[J]. Biochimica et Biophysica Acta (BBA)-General Subjects, 2017, 1861(12): 3096-3108.

[40] Duttke S H, Guzman C, Chang M, et al. Position-dependent function of human sequence-specific transcription factors[J]. Nature, 2024, 631(8022): 891-898.

[41] Engler C, Kandzia R, Marillonnet S. A one pot, one step, precision cloning method with high throughput capability[J]. PloS one, 2008, 3(11): e3647.

[42] Engler C, Marillonnet S. Golden gate cloning[J]. DNA cloning and assembly methods, 2014: 119-131.

[43] Fan H, Zhang R, Fan K, et al. Exploring the specificity of nanozymes[J]. ACS nano, 2024, 18(4): 2533-2540.

[44] Ghosh T, Nikami T. Recent development of stereoselective C-glycosylation via generation of glycosyl radical[J]. Carbohydrate Research, 2022, 522: 108677.

[45] Grasso S, Dabene V, Hendriks M M W B, et al. Signal peptide efficiency: from high-throughput data to prediction and explanation[J]. ACS synthetic biology, 2023, 12(2): 390-404.

[46] Guang W, Ding H, Czinn S J, et al. Muc1 cell surface mucin attenuates epithelial inflammation in response to a common mucosal pathogen[J]. Journal of Biological Chemistry, 2010, 285(27): 20547-20557.

[47] Guo J M, Zhang Y, Cheng L, et al. Molecular cloning and characterization of a novel member of the UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase family, pp-GalNAc-T12[J]. FEBS letters, 2002, 524(1-3): 211-218.

[48] Guo L, Zhou L, Wei P, et al. Emerging roles of UDP-GalNAc polypeptide N-acetyl -galactosaminyltransferases in cardiovascular disease[J]. Aging and Disease, 2024, 16(1): 239.

[49] Gupta G S. Animal lectins: form, function and clinical applications[M]. Springer Science & Business Media, 2012

[50] He M, Zhou X, WANG X. Glycosylation: mechanisms, biological functions and clinical implications[J]. Signal Transduction and Targeted Therapy, 2024, 9(1): 194.

[51] Hollingsworth, M.A.; Swanson, B.J. Mucins in cancer: Protection and control of the cell surface. Nat. Rev. Cancer 2004, 4, 45–60.

[52] Hu M, Zhang R, Yang J, et al. The role of N-glycosylation modification in the pathogenesis of liver cancer[J]. Cell Death & Disease, 2023, 14(3): 222.

[53] Huang J, Hou S, An J, et al. In-depth characterization of protein N-glycosylation for a COVID-19 variant-design vaccine spike protein[J]. Analytical and bioanalytical chemistry, 2023, 415(8): 1455-1464.

[54] Illiano A, Pinto G, Melchiorre C, et al. Protein glycosylation investigated by mass spectrometry: an overview[J]. Cells, 2020, 9(9): 1986.

[55] Jia F, Xiao Y, Feng Y, et al. N-glycosylation facilitates the activation of a plant cell-surface receptor[J]. Nature Plants, 2024: 1-13.

[56] Johansson M E V, Ambort D, Pelaseyed T, et al. Composition and functional role of the mucus layers in the intestine[J]. Cellular and molecular life sciences, 2011, 68: 3635-3641.

[57] Joshi R, Baek I, Joshi R, et al. Detection of fabricated eggs using Fourier transform infrared (FT-IR) spectroscopy coupled with multivariate classification techniques[J]. Infrared Physics & Technology, 2022, 123: 104163.

[58] Kasdorf B T, Weber F, Petrou G, et al. Mucin-inspired lubrication on hydrophobic surfaces[J]. Biomacromolecules, 2017, 18(8): 2454-2462.

[59] Kashyap B, Kullaa A M. Regulation of mucin 1 expression and its relationship with oral diseases[J]. Archives of Oral Biology, 2020, 117: 104791.

[60] Kaushik S, He H, Dalbey R E. Bacterial signal peptides-navigating the journey of proteins[J]. Frontiers in physiology, 2022, 13: 933153.

[61] Kumar J, Pandey A, Singh S P. An introduction to enzyme structure dynamics and enzyme catalysis[M]//Biomass, Biofuels, Biochemicals. Elsevier, 2020: 3-10.

[62] Kuribara T, Hirose M, Tajima N, et al. Chemical study of high-mannose-type glycan-related proteins to understand their functions at the molecular level[J]. Trends in Glycoscience and Glycotechnology, 2024, 36(213): E94-E103.

[63] Kuril A K, Vashi A, Subbappa P K. A comprehensive guide for secondary structure and tertiary structure determination in peptides and proteins by circular dichroism spectrometer[J]. Journal of Peptide Science, 2025, 31(1): e3648.

[64] Li J, Guo B, Zhang W, et al. Recent advances in demystifying O-glycosylation in health and disease[J]. Proteomics, 2022, 22(23-24): 2200156.

[65] Li J, Zhang J, Xu M, et al. Advances in glycopeptide enrichment methods for the analysis of protein glycosylation over the past decade[J]. Journal of Separation Science, 2022, 45(16): 3169-3186.

[66] Li M, He Y, Meng H, et al. Multiple effects of sodium dodecyl sulfate on chromogenic catalysis of tetramethylbenzidine with horseradish peroxidase[J]. Journal of Dispersion Science and Technology, 2021, 42(4): 526-536.

[67] Li W, Bilal M, Singh A K, et al. Broadening the scope of biocatalysis engineering by tailoring enzyme microenvironment: a review[J]. Catalysis Letters, 2023, 153(5): 1227-1239.

[68] Li X, Du Y, Chen X, et al. Emerging roles of O-glycosylation in regulating protein aggregation, phase separation, and functions[J]. Current Opinion in Chemical Biology, 2023, 75: 102314.

[69] Liebl K, Zacharias M. The development of nucleic acids force fields: From an unchallenged past to a competitive future[J]. Biophysical Journal, 2023, 122(14): 2841-2851.

[70] Liegeois M A, Fahy J V. The mucin gene MUC5B is required for normal lung function[J]. American Journal of Respiratory and Critical Care Medicine, 2022, 205(7): 737-739.

[71] Limonta-Fernandez M, Chinea-Santiago G, Martin-Dunn A M, et al. An engineered SARS-CoV-2 receptor-binding domain produced in Pichia pastoris as a candidate vaccine antigen[J]. New Biotechnology, 2022, 72: 11-21.

[72] Lin R, Zhang J, Xu R, et al. Developments in molecular docking technologies for application of polysaccharide-based materials: A review[J]. Critical Reviews in Food Science and Nutrition, 2024, 64(24): 8540-8552.

[73] Liu Y S, Fujita M. Mammalian GPI-anchor modifications and the enzymes involved[J]. Biochemical Society Transactions, 2020, 48(3): 1129-1138.

[74] Magalhaes A, Duarte H O, Reis C A. The role of O-glycosylation in human disease[J]. Molecular Aspects of Medicine, 2021, 79: 100964.

[75] Mahapatra S R, Dey J, Raj T K, et al. The potential of plant-derived secondary metabolites as novel drug candidates against Klebsiella pneumoniae: Molecular docking and simulation investigation[J]. South African Journal of Botany, 2022, 149: 789-797.

[76] Manne A, Esnakula A, Abushahin L, et al. Understanding the clinical impact of MUC5AC expression on pancreatic ductal adenocarcinoma[J]. Cancers, 2021, 13(12): 3059.

[77] Mao Y，Zhang Y，Fan S，et al．GALNT6 promotes tumori genicity and metastasis of breast cancer cell via β-catenin/ MUC1-C signaling pathway[J].. nternational Journal of Biological Sciences，2019，15(1) : 169～182．

[78] Markoska T, Vasiljevic T, Huppertz T. Unravelling conformational aspects of milk protein structure—contributions from nuclear magnetic resonance studies[J]. Foods, 2020, 9(8): 1128.

[79] Mcguire E J, Roseman S. Enzymatic synthesis of the protein-hexosamine linkage in sheep submaxillary mucin[J]. Journal of Biological Chemistry, 1967, 242(16): 3745-3747.

[80] Milac A L, Buchete N V, Fritz T A, et al. Substrate-induced conformational changes and dynamics of UDP-N-acetylgalactosamine: polypeptide N-acetylgalactosaminyltransferase-2[J]. Journal of molecular biology, 2007, 373(2): 439-451.

[81] Miotto M, Olimpieri P P, Di Rienzo L, et al. Insights on protein thermal stability: a graph representation of molecular interactions[J]. Bioinformatics, 2019, 35(15): 2569-2577.

[82] Mohammed A M, Khalaf Y H. Kinetic and thermodynamic study of ALP enzyme in the presence and absence MWCNTs and Pt-NPs nanocomposites[J]. Results in Chemistry, 2023, 5: 100844.

[83] Montreuil, J. Glycoproteins and Disease[J]. 1996: v.

[84] Muller G A, Muller T D. (Patho) Physiology of glycosylphosphatidylinositol-anchored proteins II: intercellular transfer of matter (inheritance?) that matters[J]. Biomolecules, 2023, 13(6): 994.

[85] Nandiyanto A B D, Ragadhita R, Fiandini M. Interpretation of Fourier transform infrared spectra (FTIR): A practical approach in the polymer/plastic thermal decomposition[J]. Indonesian Journal of Science and Technology, 2023, 8(1): 113-126.

[86] Naseri R, Navabi S J, Samimi Z, et al. Targeting Glycoproteins as a therapeutic strategy for diabetes mellitus and its complications[J]. DARU Journal of Pharmaceutical Sciences, 2020, 28: 333-358.

[87] Navaratna T, Atangcho L, Mahajan M, et al. Directed evolution using stabilized bacterial peptide display[J]. Journal of the American Chemical Society, 2019, 142(4): 1882-1894.

[88] Orlikowska M, Wyciszkiewicz A, Wegrzyn K, et al. Methods for monitoring protein-membrane binding. Comparison based on the interactions between amyloidogenic protein human cystatin C and phospholipid liposomes[J]. International Journal of Biological Macromolecules, 2024, 278: 134889.

[89] Owoloye A J, Ligali F C, Enejoh O A, et al. Molecular docking, simulation and binding free energy analysis of small molecules as Pf HT1 inhibitors[J]. PloS one, 2022, 17(8): e0268269.

[90] Palmai-pallag T, Khodabukus N, Kinarsky L,et al., Har-ris A: The role of the SEA (sea urchin sperm protein, enterokinase and agrin) module in cleavage of membrane-tethered mucins.FEBS J 2005; 272: 2901–2911.

[91] Pandey V K, Sharma R, Peajapati G K, et al. N-glycosylation, a leading role in viral infection and immunity development[J]. Molecular biology reports, 2022, 49(8): 8109-8120.

[92] Patsos G, Corfield A. O-Glycosylation: structural diversity and function[M]. Wiley-VCH: Weinheim, Germany, 2009.

[93] Piumetti M, Illanes A, Lygeros N. Molecular dynamics and complexity in catalysis and biocatalysis[M]. Cham, Switzerland: Springer International Publishing, 2022.

[94] Priya S, Tripathi G, Singh D B, et al. Machine learning approaches and their applications in drug discovery and design[J]. Chemical Biology & Drug Design, 2022, 100(1): 136-153.

[95] Pryor J M, Potapov V, Bilotti K, et al. Rapid 40 kb genome construction from 52 parts through data-optimized assembly design[J]. ACS Synthetic Biology, 2022, 11(6): 2036-2042.

[96] Psrk S, Kuo J C H, Reesink H L, et al. Recombinant mucin biotechnology and engineering[J]. Advanced drug delivery reviews, 2023, 193: 114618.

[97] Pullmann P, Knorrscheidt A, Munch J, et al. A modular two yeast species secretion system for the production and preparative application of unspecific peroxygenases[J]. Communications biology, 2021, 4(1): 562.

[98] Quesnel A, Coles N, Angione C, et al. Glycosylation spectral signatures for glioma grade discrimination using Raman spectroscopy[J]. BMC cancer, 2023, 23(1): 174.

[99] Radziejewska I. Galectin-3 and epithelial MUC1 mucin-interactions supporting cancer development[J]. Cancers, 2023, 15(10): 2680.

[100] Raghu D. GALNT3 maintains the epithelial state by promoting O-Galnac modification of e-cadherin in trophoblast stem cells and human mammary epithelial cells[M]. The University of Memphis, 2019.

[101] Riley N M, Bertozzi C R, Pitteri S J. A pragmatic guide to enrichment strategies for mass spectrometry-based glycoproteomics[J]. Molecular & Cellular Proteomics, 2021, 20.

[102] Sacanna S, Irvine W T M, Chaikin P M, et al.Lock and key colloids[J]. Nature, 2010, 464(7288): 575-578.

[103] Sahu R, Sooram B, Sasidharan S, et al. Applications of infrared spectroscopy to study proteins[M]//Advanced spectroscopic methods to study biomolecular structure and dynamics. Academic Press, 2023: 153-171.

[104] Saikia S, Bordoloi M. Molecular docking: challenges, advances and its use in drug discovery perspective[J]. Current drug targets, 2019, 20(5): 501-521.

[105] Saitou A, Hasegawa Y, Fujitani N, et al. N-glycosylation regulates MET processing and signaling[J]. Cancer Science, 2022, 113(4): 1292-1304.

[106] Sanz-Martinez I, Pereira S, Merino P, et al. Molecular recognition of GalNAc in mucin-type O-glycosylation[J]. Accounts of Chemical Research, 2023, 56(5): 548-560.

[107] Sassi P, Caponi S, Ricci M, et al. Infrared versus light scattering techniques to monitor the gel to liquid crystal phase transition in lipid membranes[J]. Journal of Raman Spectroscopy, 2015, 46(7): 644-651.

[108] Satoh T, Yagi-Utsumi M, Ishii N, et al. Structural basis of sugar recognition by SCFFBS2 ubiquitin ligase involved in NGLY1 deficiency[J]. FEBS letters, 2024, 598(18): 2259-2268.

[109] Schjoldager K T, Narimatsu Y, Joshi H J, et al. Global view of human protein glycosylation pathways and functions[J]. Nature reviews Molecular cell biology, 2020, 21(12): 729-749.

[110] Selvaraj C, Rudhra O, Alothaim A S, et al. Structure and chemistry of enzymatic active sites that play a role in the switch and conformation mechanism[J]. Advances in Protein Chemistry and Structural Biology, 2022, 130: 59-83.

[111] Sharma S, Creuzenet C, Jarrell K F, et al. Glycosyltransferase-coupled assays for 4-epimerase WbpP from pseudomonas aeruginosa[J]. Bacterial Polysaccharides: Methods and Protocols, 2019: 255-268.

[112] Spassov D S. Binding affinity determination in drug design: insights from lock and key, induced fit, conformational selection, and inhibitor trapping models[J]. International Journal of Molecular Sciences, 2024, 25(13): 7124.

[113] Spohner S C, Muller H, Quitmann H, et al. Expression of enzymes for the usage in food and feed industry with Pichia pastoris[J]. Journal of biotechnology, 2015, 202: 118-134.

[114] Srivastava A, Nagai T, Srivastava A, et al. Role of computational methods in going beyond X-ray crystallography to explore protein structure and dynamics[J]. International journal of molecular sciences, 2018, 19(11): 3401.

[115] Su D, Yao M, Liu J, et al. Enhancing mechanical properties of silk fibroin hydrogel through restricting the growth of β-sheet domains[J]. ACS applied materials & interfaces, 2017, 9(20): 17489-17498.

[116] Sun L, Zhang Y, Li W, et al. Mucin glycans: A target for cancer therapy[J]. Molecules, 2023, 28(20): 7033.

[117] Syed Z A, Zhang L, Ten Hagen K G.In vivo models of mucin biosynthesis and function[J].Adv Drug Deliv Rev,2022,184:114182.

[118] Szent-Gyorgyi C, Perkins L A, Schmidt B F, et al. Bottom-Up design: a modular golden gate assembly platform of yeast plasmids for simultaneous secretion and surface display of distinct FAP fusion proteins[J]. ACS Synthetic Biology, 2022, 11(11): 3681-3698.

[119] Tatusova T, DiCuccio M, Badretdin A, et al. NCBI prokaryotic genome annotation pipeline[J]. Nucleic acids research, 2016, 44(14): 6614-6624.

[120] Taus C, Windwarder M, Altmann F, et al. UDP-N-acetyl-α-D-galactosamine: polypeptide N-acetylgalactosaminyl-transferase from the snail biomphalaria glabrata-substrate specificity and preference of glycosylation sites[J]. Glycoconjugate Journal, 2014, 31: 661-670.

[121] Tenno M, Ohtsubo K, Hagen F K, et al. Initiation of protein O glycosylation by the polypeptide GalNAcT-1 in vascular biology and humoral immunity[J]. Molecular and cellular biology, 2007, 27(24): 8783-8796.

[122] Thaysen-Andersen M , Packer N H .Advances in LC-MS/MS-based glycoproteomics : getting closer to system-wide site-specific mapping of the N- and O-glycoproteome[J].Biochimica Et Biophysica Acta, 2014, 1844(9):1437-1452.

[123] Thompson N, Wakarchuk W. O-glycosylation and its role in therapeutic proteins[J]. Bioscience Reports, 2022, 42(10): BSR20220094.

[124] Tinti M, Ferguson M A J. Visualisation of experimentally determined and predicted protein N-glycosylation and predicted glycosylphosphatidylinositol anchor addition in Trypanosoma brucei[J]. Wellcome Open Research, 2022, 7.Van der Zee J. The Gate: The true story of the design and construction of the Golden Gate Bridge[M]. Plunkett Lake Press, 2024

[125] Van Putten J P M, Strijbis K. Transmembrane mucins: signaling receptors at the intersection of inflammation and cancer[J]. Journal of innate immunity, 2017, 9(3): 281-299.

[126] Verzijl C R C, Oldoni F, Loaiza N, et al. A novel role for GalNAc-T2 dependent glycosylation in energy homeostasis[J]. Molecular metabolism, 2022, 60: 101472.

[127] Voronina L, Fleischmann F, Simunovic J, et al. Probing blood plasma protein glycosylation with infrared spectroscopy[J]. Analytical Chemistry, 2024, 96(7): 2830-2839.

[128] Wang Y, Liu L, Yu Y. Mucins and mucinous ovarian carcinoma: Development, differential diagnosis, and treatment[J]. Heliyon, 2023.

[129] Webb B, Sali A. Protein structure modeling with MODELLER[J]. Functional genomics: Methods and protocols, 2017: 39-54.

[130] Wu G, Kim D, Kim J N, et al. A Mucin1 C-terminal subunit-directed monoclonal antibody targets overexpressed Mucin1 in breast cancer[J]. Theranostics, 2018, 8(1): 78.

[131] Wu Q, Xie X, Zhang K, et al. Reduced expression of ppGalNAc-T4 promotes proliferation of human breast cancer cells[J]. Cell Biology International, 2021, 45(2): 320-333.

[132] Wu X, Ren Y, Chen S, et al. Production of L-lactic acid from methanol by engineered yeast Pichia pastoris[J]. Bioresource Technology, 2025, 415: 131730.

[133] Xiao G, Jiang Z, Xing T, et al. Small ubiquitin-like modifier protease gene TaDSU enhances salt tolerance of wheat[J]. New Phytologist, 2024.

[134] Xie Z, He J, Peng S, et al. Biosynthesis of protein-based drugs using eukaryotic microalgae[J]. Algal Research, 2023, 74: 103219.

[135] Xu Y, Jia G, Li T, et al. Molecular insights into biogenesis of glycosylphosphatidylinositol anchor proteins[J]. Nature Communications, 2022, 13(1): 2617.

[136] Yamamoto D, Hongo H, Kosaka T, et al. The sialyl-Tn antigen synthase genes regulates migration–proliferation dichotomy in prostate cancer cells under hypoxia[J]. Glycoconjugate Journal, 2023, 40(2): 199-212.

[137] Yan X, Liu X, Zhao C, et al. Applications of synthetic biology in medical and pharmaceutical fields[J]. Signal transduction and targeted therapy, 2023, 8(1): 199.

[138] Yang S, Zhang Q, Yang H, et al. Progress in infrared spectroscopy as an efficient tool for predicting. protein secondary structure[J]. International Journal of Biological Macromolecules, 2022, 206: 175-187.

[139] Yang W, Tian E, Chernish A, et al. Quantitative mapping of the in vivo O-GalNAc glycoproteome in mouse tissues identifies GalNAc-T2 O-glycosites in metabolic disorder[J]. Proceedings of the National Academy of Sciences, 2023, 120(43): e2303703120.

[140] Zhai X, Yang R, Chu Q, et al. AMPK-regulated glycerol excretion maintains metabolic crosstalk between reductive and energetic stress[J]. Nature Cell Biology, 2025: 1-13.

[141] Zhang C, Zhang X, Freddolino P L, et al. BioLiP2: an updated structure database for biologically relevant ligand–protein interactions[J]. Nucleic acids research, 2024, 52(D1): D404-D412.

[142] Zhang Q, Kobras C M, Gebhard S, et al. Regulation of heterologous subtilin production in Bacillus subtilis W168[J]. Microbial Cell Factories, 2022, 21(1): 57.

[143] Zhang Q, Li L, Lan Q, et al. Protein glycosylation: A promising way to modify the functional properties and extend the application in food system[J]. Critical reviews in food science and nutrition, 2019, 59(15): 2506-2533.

[144] Zhao Y, Li M, Mao J, et al. Immunomodulation of wound healing leading to efferocytosis[J]. Smart Medicine, 2024, 3(1): e2023003