GlycoBIST: A System for Automatic Glycan Profiling of a Target Protein Using Milli-Bead Array in a Tip.

Lectin is a biomolecule that recognizes a specific part of glycans and, thus, has been used widely as a probe for glycoprotein analysis. Owing to the wide repertoire in nature combined with the recent two decades of advances in microarray technology, the multiplexed use of lectins has been widely used for glycan profiling of endogenous proteins. Because protein glycosylation is recognized as being biologically important and is expected to be a reliable disease marker, lectin microarray analysis with highly sensitive detection has been used to discover disease-relevant glycosylation alterations.

Male-specific cardiac pathologies in mice lacking either the A or B subunit of factor XIII.

Factor XIII (FXIII) is a proenzyme of plasma transglutaminase consisting of enzymatic A subunits (FXIII-A) and non-catalytic B subunits (FXIII-B), and acts in haemostasis and wound healing. We generated mice lacking either FXIII-A or FXIII-B to investigate the physiological functions of FXIII in vivo. A longitudinal study was carried out using the gene-targeted mice to explore the possible effects of FXIII deficiency on aging.

Administration of factor XIII B subunit increased plasma factor XIII A subunit levels in factor XIII B subunit knock-out mice.

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.

Development of a lectin microarray based on an evanescent-field fluorescence principle.

To investigate protein-carbohydrate interactions in a comprehensive and high-throughput manner, carbohydrate biosensors including microarrays have recently attracted increased attention. In this context, carbohydrate and lectin microarrays are emerging as techniques to meet such requisites. However, most of these methods adopt a conventional immuno-detection system, which requires repetitive washing steps before detection.

Application of lectin microarray to crude samples: differential glycan profiling of lec mutants.

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.

Evanescent-field fluorescence-assisted lectin microarray: a new strategy for glycan profiling.

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.

R255h amino acid substitution of protein Z identified in patients with factor V Leiden mutation.

The clinical significance of diminished protein Z in plasma is controversial. Studies in mice demonstrated that deficiency of protein Z dramatically increases the prothrombotic tendency of factor V Leiden mutation. This finding was confirmed by initial results in humans, indicating that thromboembolism in factor V Leiden patients with lowered protein Z level occurs earlier than in patients with normal protein Z levels.

A naturally occurring E30Q mutation in the Gla domain of protein Z causes its impaired secretion and subsequent deficiency.

Protein Z is a vitamin K-dependent glycoprotein that plays a role in the regulation of coagulation. A nucleotide substitution of G by C in exon II of the protein Z gene, resulting in the replacement of Glu-30 with Gln (E30Q), and a G to A transition at the 79th nucleotide in intron F (IntF79G/A) were heterozygously identified in a patient with a severe thrombotic tendency, whose plasma protein Z level was about 15% of normal. Other vitamin K-dependent coagulation factors were within normal ranges.

Factor XIII A subunit-deficient mice developed severe uterine bleeding events and subsequent spontaneous miscarriages.

To understand the molecular pathology of factor XIII (FXIII) deficiency in vivo, its A subunit (FXIIIA)-knockout (KO) mice were functionally analyzed. Although homozygous FXIIIA female KO mice were capable of becoming pregnant, most of them died due to excessive vaginal bleeding during gestation. Abdominal incisions revealed that the uteri of the dead mice were filled with blood and that some embryos were much smaller than others within a single uterus.