Molecular Biology, Rowan Virtua SOM, Rowan University, 2 Medical Center Dr., Stratford, NJ, 08084, USA.
Rutgers New Jersey Medical School, 185 S Orange Ave, Newark, NJ, 07103, USA.
Biochem Biophys Res Commun. 2024 May 28;710:149881. doi: 10.1016/j.bbrc.2024.149881. Epub 2024 Apr 3.
Maackia amurensis lectins serve as research and botanical agents that bind to sialic residues on proteins. For example, M. amurensis seed lectin (MASL) targets the sialic acid modified podoplanin (PDPN) receptor to suppress arthritic chondrocyte inflammation, and inhibit tumor cell growth and motility. However, M. amurensis lectin nomenclature and composition are not clearly defined. Here, we sought to definitively characterize MASL and its effects on tumor cell behavior. We utilized SDS-PAGE and LC-MS/MS to find that M. amurensis lectins can be divided into two groups. MASL is a member of one group which is composed of subunits that form dimers, evidently mediated by a cysteine residue in the carboxy region of the protein. In contrast to MASL, members of the other group do not dimerize under nonreducing conditions. These data also indicate that MASL is composed of 4 isoforms with an identical amino acid sequence, but unique glycosylation sites. We also produced a novel recombinant soluble human PDPN receptor (shPDPN) with 17 threonine residues glycosylated with sialic acid moieties with potential to act as a ligand trap that inhibits OSCC cell growth and motility. In addition, we report here that MASL targets PDPN with very strong binding kinetics in the nanomolar range. Moreover, we confirm that MASL can inhibit the growth and motility of human oral squamous cell carcinoma (OSCC) cells that express the PDPN receptor. Taken together, these data characterize M. amurensis lectins into two major groups based on their intrinsic properties, clarify the composition of MASL and its subunit isoform sequence and glycosylation sites, define sialic acid modifications on the PDPN receptor and its ability to act as a ligand trap, quantitate MASL binding to PDPN with KD in the nanomolar range, and verify the ability of MASL to serve as a potential anticancer agent.
马卡西亚蓖麻凝集素是一种研究和植物学试剂，能够与蛋白质上的唾液酸残基结合。例如，马卡西亚蓖麻种子凝集素（MASL）靶向唾液酸化的 podoplanin（PDPN）受体，抑制关节炎软骨细胞炎症，并抑制肿瘤细胞生长和运动。然而，马卡西亚蓖麻凝集素的命名法和组成尚不清楚。在这里，我们试图明确地描述 MASL 及其对肿瘤细胞行为的影响。我们利用 SDS-PAGE 和 LC-MS/MS 发现，马卡西亚蓖麻凝集素可以分为两组。MASL 是一组的成员，由形成二聚体的亚基组成，显然由蛋白质羧基区域的半胱氨酸残基介导。与 MASL 不同，另一组的成员在非还原条件下不二聚化。这些数据还表明，MASL 由 4 种具有相同氨基酸序列但独特糖基化位点的同工型组成。我们还生产了一种新型重组可溶性人 PDPN 受体（shPDPN），其 17 个苏氨酸残基被唾液酸部分糖基化，具有作为配体陷阱的潜力，可以抑制口腔鳞状细胞癌（OSCC）细胞的生长和运动。此外，我们在这里报告 MASL 以纳米摩尔范围内的非常强的结合动力学靶向 PDPN。此外，我们证实 MASL 可以抑制表达 PDPN 受体的人口腔鳞状细胞癌细胞的生长和运动。总之，这些数据根据其内在特性将马卡西亚蓖麻凝集素分为两大主要组，阐明了 MASL 的组成及其亚基同工型序列和糖基化位点，定义了 PDPN 受体上的唾液酸修饰及其作为配体陷阱的能力，定量了 MASL 与 PDPN 的结合，KD 值在纳摩尔范围内，并验证了 MASL 作为潜在抗癌剂的能力。
