Glycans have important roles in living organisms with their structural diversity. Thus, glycomics, especially aspects involving the assignment of functional glycans in a high-throughput manner, has been an emerging field in the postproteomics era. To date, however, there has been no versatile method for glycan profiling. Here we describe a new microarray procedure based on an evanescent-field fluorescence-detection principle, which allows sensitive, real-time observation of multiple lectin-carbohydrate interactions under equilibrium conditions. The method allows quantitative detection of even weak lectin-carbohydrate interactions (dissociation constant, K(d) > 10(-6) M) as fluorescent signals for 39 immobilized lectins. We derived fully specific signal patterns for various Cy3-labeled glycoproteins, glycopeptides and tetramethylrhodamine (TMR)-labeled oligosaccharides. The obtained results were consistent with the previous reports of glycoprotein and lectin specificities. We investigated the latter aspects in detail by frontal affinity chromatography, another profiling method. Thus, the developed lectin microarray should contribute to creation of a new paradigm for glycomics.
IntroductionCoagulation factor XIII (FXIII) is a heterotetramer consisting of A subunits (FXIIIA) and B subunits (FXIIIB). FXIII catalyzes intermolecular cross-linking reactions between fibrin monomers, ␣ 2 -plasmin inhibitor, and fibronectin. These reactions increase the mechanical strength of the fibrin clot and its resistance to proteolytic degradation and enhance the assembly of the extracellular matrix. Accordingly, a deficiency of FXIII results in defective cross-linking reactions of these substrates and is thus associated with a number of diseases. 1 Congenital FXIII deficiency is caused by defects in the F13A or F13B genes, 2 leading to a bleeding tendency and abnormal wound healing in affected patients and spontaneous miscarriage in female patients. [3][4][5] To understand the molecular pathology of these deficiencies, we have identified a number of mutations in the F13A and F13B genes in patients' DNA and have analyzed the molecular mechanisms of FXIII deficiency using in vitro procedures. 6-15 Because it is not possible to understand completely the clinical pathologic mechanisms of this disease in vivo, we performed functional analyses on FXIIIA knockout (KO) mice. Study designFXIIIA KO mice were created by replacing exon VII with a neomycinresistance cassette as described previously. 16 All KO and wild-type CBA mice were housed in a specific pathogen-free facility. Experimental procedures were approved by the Animal Care and Use Committee of Yamagata University and were carried out in accordance with the guidelines of this committee and that of Japanese governmental law.Using tail biopsy DNA, genotyping of the mice was performed by polymerase chain reaction (PCR) with 2 forward primers designed from the neomycin-resistant gene (5Ј-CAC TGC ATT CTA GTT GTG GTT TGT CC-3Ј) and exon VII of the mouse F13A gene (5Ј-GCC AAG GAT GAT GAA GGT GTT CTT-3Ј), respectively, and a common reverse primer from intron G (5Ј-CCC TGA GAC TTA CGG ATG AAG AAG-3Ј).Tissues collected for histologic analyses were placed in 10% neutralbuffered formalin, processed into paraffin, and 3-m sections were prepared and stained with hematoxylin and eosin by standard techniques.The distribution of events between the study groups was analyzed using 4 ϫ 2 contingency tables for individual study groups as well as 2 ϫ 2 tables for a combined group of female wild-type mice and female homozygous FXIIIA KO mice. The 2 test was also carried out by using StatView software (Abacus Concepts, Berkeley, CA). Differences were considered statistically significant at P values less than .05. Results and discussionWestern blot analysis and amine-incorporation enzymatic assay confirmed the complete absence of FXIIIA in the uterus and plasma (where FXIIIA is normally expressed/present) of the homozygous FXIIIA KO mice identified by genotyping (data not shown). Despite the complete loss of FXIIIA, all homozygous FXIIIA KO mice had appeared normal at birth as previously reported. 16 Whereas all 20 of the female homozygous KO mice survived, 3 of 20 male KO mice...
We recently developed a novel system for lectin microarray based on the evanescent-field fluorescence-detection principle, by which even weak lectin-oligosaccharide interactions are detectable without a washing procedure. For its practical application, cell glycan analysis was performed for Chinese hamster ovary (CHO) cells and their glycan profile was compared with those of their glycosylation-defective Lec mutants. Each of the cell surface extracts gave a significantly different profile from that of the parental CHO cells in a manner reflecting denoted biosynthetic features. Hence, the developed lectin microarray system is considered to be fully applicable for differential glycan profiling of crude samples.
Factor XIII (FXIII) is a proenzyme of plasma transglutaminase consisting of enzymatic A (FXIII-A) and noncatalytic B subunits (FXIII-B), and acts in hemostasis and wound healing. We freshly generated mice lacking either FXIII-A or FXIII-B to investigate the physiological functions of FXIII in vivo. Mice carrying the disrupted allele were born at the expected Mendelian ratios, and the homozygous mice were viable and fertile under specific pathogen-free conditions. Although all homozygous and heterozygous mice showed no marked difference from the wild-type animals in general appearance, homozygous mice of either FXIII-A- or FXIII-B-deficiency did have prolonged bleeding times. It was confirmed that thrombin-dependent amine incorporation and fibrin-crosslinking in plasma were undetectable in the FXIII-A-deficient mice and markedly reduced in the FXIII-B-deficient mice; however, the gene expression of each subunit was regulated independently. Recombinant human FXIII-B (rFXIII-B) was expressed in a baculovirus expression system. When rFXIII-B was injected into FXIII-B-deficient mice, FXIII-A levels, fibrin crosslinking, and amine-incorporation activities increased in their plasma, indicating that FXIII-B assisted the maintenance of FXIII-A levels in the circulation. These mouse strains will be useful in exploring the possible pathophysiological roles of each subunit in vivo.
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