Cell surface glycans and oligosaccharides attached to proteins play important tasks in cell-cell relationships and biological functions of the cell and glycoproteinsin vivo. japonicalectin bound specifically to EBC-1 and HEK293 cells while otherWisterialectins bound equally to all of the cell lines tested.Wisteria japonicalectin binding to EBC-1 and HEK293 cells was dramatically decreased in the presence ofN-acetylgalactosamine, but not galactose, mannose, orN-acetylglucosamine, and was completely abrogated by -hexosaminidase-digestion of these cells. These results clearly demonstrate thatWisteria japonicalectin binds to terminalN-acetylgalactosamine but not galactose. In addition, histochemical analysis of human being squamous cell carcinoma cells sections shown thatWisteria japonicalectin specifically bound to differentiated malignancy tissues but not normal tissue. This novel binding characteristic ofWisteria japonicalectin has the potential to become a powerful tool for medical applications. == Intro == Many types of lectins, which are defined as p75NTR proteins having the ability to bind sugars, have been isolated from bacteria, plant and animals. Cell surface glycans and oligosaccharides attached to proteins play important tasks in cell-cell relationships and biological functions of the cell and glycoproteinsin vivo. Glycoproteins in the blood and pathological cells regularly possess unusual glycan constructions. To investigate the significance of unique glycosylation states, it is crucial to identify lectins that discriminate between unique and stringent sugars constructions among many kinds of oligosaccharides. Wisteria japonicawas 1st introduced to Europe by Phililpp von Siebold in 1830 collectively withWisteria floribundaandWisteria brachybotrys[1]. At that time,Wisteria japonicahad been placed in the genusMillettiabased within the terminal racemes inWisteriaas opposed to lateral racemes inMillettia[1] and the absence of a pair of thickened callosities, which differed from otherWisteriaspecies [1]. However, recent sequence studies of chloroplast and nuclear DNA have shown that theWisteria japonicabelongs in the genusWisteriarather thanMillettia[14]. Wisteria floribunda agglutinin (WFA) has been studied in detail and is reported to have unique biological activities, including hemagglutinating capacity and the induction of lymphocyte activation [57]. WFA has a higher affinity forN-acetylgalactosaminides than for galactosides [8], and subsequent study shown that it could bind to GalNAc1-4GlcNAc [9], whose sugars sequence was first observed within the pituitary glycoprotein hormones, lutropin (LH), thyrotropin (TSH) and follitropin (FSH) [10,11]. However, this sugar structure is the most uncommon constituent of glycans on vertebrate proteins. The GalNAc1-4GlcNAc sequence of oligosaccharides is definitely formed by the activity of 1 1,4-N-acetylgalactosaminyltransferases, and such transferases are hardly ever indicated in unique cells and organs [12,13]. Recently, GalNAc1-4GlcNAc changes ofN-glycans has been well recorded [14,15]; however, the implication of this novel modification with respect to the posttranslational control of protein function remains unfamiliar because of the lack of a specific probe for the GalNAc1-4GlcNAc sequence. In the present study, we purified a novel leguminous lectin fromWisteria japonicaseeds whose specificity is definitely specific for GalNAc1-4GlcNAc and GalNAc1-3GlcNAc sequences. Interestingly, theWisteria japonicalectin (WJA) strongly bound to EBC-1 human being squamous cell carcinoma cells and specifically stained the cancerous portions T0901317 of lung specimens from lung squamous cell carcinoma individuals. == Materials and Methods == == Preparation ofWisteria lectins == Wisteriaseeds (Wisteria floribunda, Wisteria brachybotrys, andWisteria japonica) were purchased from Unique Vegetation Co. (Tateyama, Japan) and classification of the seeds were confirmed by the help of Dr. Jin Murata (Koishikawa Botanical Landscapes, The University or college of Tokyo). Purification ofWisterialectins was performed according to the method of Toyoshima et T0901317 al. [5] with small modifications. Briefly, finely powderedWisteriaseeds were suspended in 10 mM phosphate buffer (pH 7.4) containing 0.15 M NaCl (PBS) and stirred at 4C for 18 h. After centrifugation at 17,000 gfor 1 h, obvious supernatant was combined with (NH4)2SO4to give 80% saturation. The precipitated portion was acquired by centrifugation, resuspended in distilled water and dialyzed against 50 mM phosphate buffer (pH 5.0). Lectin fractions were purified by cationic ion exchange chromatography on a Toyopearl SP-550C column (Toso, Tokyo, Japan) followed by gel filtration chromatography on a HiLoad 26/60 Superdex 200 column (prep grade, GE Healthcare, Buckinghamshire, UK) using the AKTA Explorer system (GE Healthcare). The activity of lectin was monitored by hemagglutination using sialidase (Nacalai Tesque, Kyoto, Japan)-treated mouse reddish blood cells. The purity of the lectin was checked by SDS polyacrylamide gel electrophoresis according to the method of Laemmli. Purified lectin fractions were dialyzed against distilled water and lyophilized. The N-terminal amino acid sequences of the purified lectins were analyzed by a Procise 492cLC protein sequencer (Applied Biosystems, Foster City, CA). == Glycan T0901317 microarray == The sugar-binding specificity ofWisterialectins was analyzed from the glycan microarray explained in detail inFigure S1[16].Wisterialectins were labeled with Cy3-N-hydroxysuccinimide ester (NHS-Cy3, GE Healthcare) while described previously [16]. After eliminating excess amounts of NHS-Cy3 by gel filtration on a Sephadex G-25 column (GE Healthcare), Cy3-labeledWisterialectins (5 g/ml) inside a probing buffer [25 mM Tris-HCl (pH 7.4), 0.15 M NaCl,.
