Prakobphol A, Thomsson K A, Hansson G C, Rosen S D, Singer M S, Phillips N J, Medzihradszky K F, Burlingame A L, Leffler H, Fisher S J
Department of Stomatology, University of California, San Francisco, San Francisco, California 94143, USA.
Biochemistry. 1998 Apr 7;37(14):4916-27. doi: 10.1021/bi972612a.
Previously we showed that the low-molecular-weight mucin (MG2, encoded by MUC7), a major component of human submandibular/sublingual saliva, is a bacterial receptor that coats the tooth surface. Here we tested the hypothesis that the structure of its carbohydrate residues contains important information about its function. Purified MG2 (Mr 120 000) was digested with trypsin, and the resulting Mr 90 000 fragment, which carried primarily O-linked oligosaccharides, was subjected to reductive beta-elimination. The released oligosaccharides were characterized by using nuclear magnetic resonance spectroscopy and mass spectrometry. Of the 41 different structures we detected, the most prominent included NeuAcalpha2-->3Galbeta1-->3GalNAc-ol (sialyl-T antigen), Galbeta1-->4(Fucalpha1-->3)GlcNAcbeta1-->6(Galbeta1 -->3)GalNAc-ol [type 2 core with Lewisx (Lex) determinant], and NeuAcalpha2-->3Galbeta1-->4(Fucalpha1-->3)GlcNAcbet a1-->6(Galbeta1--> 3) GalNAc-ol [type 2 core with sialyl Lex (sLex) determinant]. We also detected di-, tri-, and pentasaccharides with one sulfate group. Lex, sLex, and related sulfated structures are ligands for selectins, adhesion molecules that mediate leukocyte trafficking. Therefore, we investigated whether MG2 was a selectin ligand. In an enzyme-linked immunosorbent assay, L-selectin chimeras interacted with immobilized MG2 in a Ca2+-dependent manner. L-Selectin chimeras also bound to MG2 immobilized on nitrocellulose. Together, these results suggest that the saccharides that MG2 carries could specify some of its important functions, which may include mediating leukocyte interactions in the oral cavity.
此前我们发现，人下颌下腺/舌下腺唾液的主要成分——低分子量粘蛋白（由MUC7编码的MG2）是一种覆盖牙齿表面的细菌受体。在此，我们检验了这样一个假说，即其碳水化合物残基的结构包含有关其功能的重要信息。用胰蛋白酶消化纯化的MG2（分子量120 000），得到的主要带有O-连接寡糖的分子量90 000片段进行还原性β-消除反应。释放出的寡糖通过核磁共振光谱和质谱进行表征。在我们检测到的41种不同结构中，最主要的包括NeuAcalpha2-->3Galbeta1-->3GalNAc-ol（唾液酸-T抗原）、Galbeta1-->4(Fucalpha1-->3)GlcNAcbeta1-->6(Galbeta1 -->3)GalNAc-ol [带有Lewisx（Lex）决定簇的2型核心结构]，以及NeuAcalpha2-->3Galbeta1-->4(Fucalpha1-->3)GlcNAcbet a1-->6(Galbeta1--> 3) GalNAc-ol [带有唾液酸Lex（sLex）决定簇的2型核心结构]。我们还检测到带有一个硫酸基团的二糖、三糖和五糖。Lex、sLex及相关的硫酸化结构是选择素的配体，选择素是介导白细胞迁移的黏附分子。因此，我们研究了MG2是否为一种选择素配体。在酶联免疫吸附测定中，L-选择素嵌合体以Ca2+依赖的方式与固定化的MG2相互作用。L-选择素嵌合体也与固定在硝酸纤维素上的MG2结合。总之，这些结果表明，MG2携带的糖类可能决定其一些重要功能，其中可能包括介导口腔中的白细胞相互作用。
