In normal cells, glycan binding proteins mediate various cellular processes upon recognition and binding to respective ligands. In tumor cells, these proteins have been associated with metastasis. Lactosyl-sepharose binding proteins (LSBPs) were isolated and identified in a workflow involving lactosyl affinity chromatography and label-free quantification mass spectrometry (LFQ MS). A binding study with monosaccharides was performed by microscale thermophoresis and nuclear magnetic resonance spectroscopy. Influence of galactose on LSBPs’ binding to the lactosyl resin was investigated by competitive affinity chromatography followed by LFQ MS. An analysis of amino acids with sugar binding motifs was searched using bioinformatics tools. The expression profiles of these proteins at the mRNA level, as determined by a chip array from a pancreatic ductal adenocarcinoma (PDAC) liver metastasis model, were used for evaluating their potential role in cancer progression. Proteomics data and their respective genes were analyzed by MaxQuant and Ingenuity Pathway Analysis. In total, 1295 LSBPs were isolated and identified from Suit2-007 human pancreatic adenocarcinoma cells. Interaction studies revealed that these proteins exhibit low to moderate affinity for monosaccharide sugars. Some of these LSBPs even showed reduced affinity after calcium depletion. Among the isolated proteins were annexins and galectins in addition to other families, with no history of binding lactosyl residues. A subset of LSBPs exhibited differential profiles in the pancreas, liver, and lung environments. These modulations may be related to tumor progression. In conclusion, we show that PDAC cells contain LSBPs, a subset of which binds galactose with calcium dependency. The differential expression of these proteins in a rat model highlights their value for diagnosis and as potential drug targets for PDAC therapy. Future work will be required to validate these findings in patient samples.

Impact statement

Interaction of glycan binding proteins with aberrantly expressed glycans in tumor environment is crucial for metastasis. Here, we established a work flow for investigating the presence of a subset of these proteins in PDAC cells, which bind to a lactosyl-sepharose resin. The resin had been designed to isolate proteins with lectin-like properties. The corresponding lactosyl-sepharose binding proteins (LSBPs) show affinity for galactose and other monosaccharides. A subset of the LSBPs shows also calcium dependency. The importance of these proteins is highlighted by their differential expression profiles in PDAC cells growing in primary (pancreas) and metastatic (liver and lung) organ sites. Based on their affinity for the lactosyl-resin and monosaccharides, LSBPs hold potential for PDAC diagnosis and as drug targets. This work has set the stage for further investigation of the occurrence and the role of LSBPs in patient samples using the newly established workflow.

中文翻译：

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来自胰腺癌细胞的乳糖基-琼脂糖结合蛋白在原发和转移器官中显示差异表达。

在正常细胞中，聚糖结合蛋白在识别并结合各自的配体后介导各种细胞过程。在肿瘤细胞中，这些蛋白质与转移有关。分离乳糖基-琼脂糖结合蛋白（LSBPs），并在涉及乳糖基亲和色谱和无标记定量质谱（LFQ MS）的工作流程中进行鉴定。通过微尺度热泳和核磁共振波谱进行了与单糖的结合研究。通过竞争性亲和色谱，然后进行LFQ MS，研究了半乳糖对LSBP与乳糖基树脂结合的影响。使用生物信息学工具搜索具有糖结合基序的氨基酸的分析。这些蛋白质在mRNA水平上的表达谱，如由胰管腺癌（PDAC）肝转移模型的芯片阵列所确定的，用于评估其在癌症进展中的潜在作用。通过MaxQuant和Ingenuity Pathway Analysis分析蛋白质组学数据及其各自的基因。总共从Suit2-007人胰腺腺癌细胞中分离并鉴定了1295个LSBP。相互作用研究表明，这些蛋白质对单糖的亲和力较低。这些LSBP中的一些甚至在钙耗尽后显示出降低的亲和力。除其他家族外，分离的蛋白质还有膜联蛋白和半乳凝素，没有结合乳糖基残基的历史。LSBP的一个子集在胰腺，肝脏和肺部环境中表现出差异特征。这些调节可能与肿瘤进展有关。结论，我们显示PDAC细胞包含LSBP，其子集结合具有钙依赖性的半乳糖。这些蛋白质在大鼠模型中的差异表达突出了其在诊断中的价值，并作为PDAC治疗的潜在药物靶标。将需要进一步的工作来验证患者样本中的这些发现。

影响陈述

聚糖结合蛋白与肿瘤环境中异常表达的聚糖的相互作用对于转移至关重要。在这里，我们建立了一个工作流程，用于研究PDAC细胞中这些蛋白质的一个子集的存在，该子集与乳糖基-琼脂糖树脂结合。该树脂被设计用来分离具有凝集素样特性的蛋白质。相应的乳糖基-琼脂糖结合蛋白（LSBP）显示出对半乳糖和其他单糖的亲和力。LSBP的子集也显示出钙依赖性。这些蛋白质的重要​​性通过在原发（胰腺）和转移性（肝和肺）器官部位生长的PDAC细胞中的差异表达谱突显出来。基于它们对乳糖基树脂和单糖的亲和力，LSBPs具有用于PDAC诊断和作为药物靶标的潜力。