A von Willebrand factor fragment containing the D'D3 domains is sufficient to stabilize coagulation factor VIII in mice.

Plasma factor VIII (FVIII) and von Willebrand factor (VWF) circulate together as a complex. We identify VWF fragments sufficient for FVIII stabilization in vivo and show that hepatic expression of the VWF D'D3 domains (S764-P1247), either as a monomer or a dimer, is sufficient to raise FVIII levels in Vwf(-/-) mice from a baseline of ∼5% to 10%, to ∼50% to 100%. These results demonstrate that a fragment containing only ∼20% of the VWF sequence is sufficient to support FVIII stability in vivo. Expression of the VWF D'D3 fragment fused at its C terminus to the Fc segment of immunoglobulin G1 results in markedly enhanced survival in the circulation (t1/2 > 7 days), concomitant with elevated plasma FVIII levels (>25% at 7 days) in Vwf(-/-) mice. Although the VWF D'D3-Fc chimera also exhibits markedly prolonged survival when transfused into FVIII-deficient mice, the cotransfused FVIII is rapidly cleared. Kinetic binding studies show that VWF propeptide processing of VWF D'D3 fragments is required for optimal FVIII affinity. The reduced affinity of VWF D'D3 and VWF D'D3-Fc for FVIII suggests that the shortened FVIII survival in FVIII-deficient mice transfused with FVIII and VWF D'D3/D'D3-Fc is due to ineffective competition of these fragments with endogenous VWF for FVIII binding.

Expression of the blood-group-related glycosyltransferase B4galnt2 influences the intestinal microbiota in mice.

Glycans on mucosal surfaces have an important role in host-microbe interactions. The locus encoding the blood-group-related glycosyltransferase ß-1,4-N-acetylgalactosaminyltransferase 2 (B4galnt2) is subject to strong selective forces in natural house-mouse populations that contain a common allelic variant that confers loss of B4galnt2 gene expression in the gastrointestinal (GI) tract. We reasoned that altered glycan-dependent intestinal host-microbe interactions may underlie these signatures of selection. To determine whether B4galnt2 influences the intestinal microbial ecology, we profiled the microbiota of wild-type and B4galnt2-deficient siblings throughout the GI tract using 16S rRNA gene pyrosequencing. This revealed both distinct communities at different anatomic sites and significant changes in composition with respect to genotype, indicating a previously unappreciated role of B4galnt2 in host-microbial homeostasis. Among the numerous B4galnt2-dependent differences identified in the abundance of specific bacterial taxa, we unexpectedly detected a difference in the pathogenic genus, Helicobacter, suggesting Helicobacter spp. also interact with B4galnt2 glycans. In contrast to other glycosyltransferases, we found that the host intestinal B4galnt2 expression is not dependent on presence of the microbiota. Given the long-term maintenance of alleles influencing B4galnt2 expression by natural selection and the GI phenotypes presented here, we suggest that variation in B4galnt2 GI expression may alter susceptibility to GI diseases such as infectious gastroenteritis.

Selection on cis-regulatory variation at B4galnt2 and its influence on von Willebrand factor in house mice.

The RIIIS/J inbred mouse strain is a model for type 1 von Willebrand disease (VWD), a common human bleeding disorder. Low von Willebrand factor (VWF) levels in RIIIS/J are due to a regulatory mutation, Mvwf1, which directs a tissue-specific switch in expression of a glycosyltransferase, B4GALNT2, from intestine to blood vessel. We recently found that Mvwf1 lies on a founder allele common among laboratory mouse strains. To investigate the evolutionary forces operating at B4galnt2, we conducted a survey of DNA sequence polymorphism and microsatellite variation spanning the B4galnt2 gene region in natural Mus musculus domesticus populations. Two divergent haplotypes segregate in these natural populations, one of which corresponds to the RIIIS/J sequence. Different local populations display dramatic differences in the frequency of these haplotypes, and reduced microsatellite variability near B4galnt2 within the RIIIS/J haplotype is consistent with the recent action of natural selection. The level and pattern of DNA sequence polymorphism in the 5' flanking region of the gene significantly deviates from the neutral expectation and suggests that variation in B4galnt2 expression may be under balancing selection and/or arose from a recently introgressed allele that subsequently increased in frequency due to natural selection. However, coalescent simulations indicate that the heterogeneity in divergence between haplotypes is greater than expected under an introgression model. Analysis of a population where the RIIIS/J haplotype is in high frequency reveals an association between this haplotype, the B4galnt2 tissue-specific switch, and a significant decrease in plasma VWF levels. Given these observations, we propose that low VWF levels may represent a fitness cost that is offset by a yet unknown benefit of the B4galnt2 tissue-specific switch. Similar mechanisms may account for the variability in VWF levels and high prevalence of VWD in other mammals, including humans.