Absence of Post-translational Aspartyl β-Hydroxylation of Epidermal Growth Factor Domains in Mice Leads to Developmental Defects and an Increased Incidence of Intestinal Neoplasia

Abstract

The BAH genomic locus encodes three distinct proteins: junctin, humbug, and BAH. All three proteins share common exons, but differ significantly based upon the use of alternative terminal exons. The biological roles of BAH and humbug and their functional relationship to junctin remain unclear. To evaluate the role of BAH in vivo, the catalytic domain of BAH was specifically targeted such that the coding regions of junctin and humbug remained undisturbed. BAH null mice lack measurable BAH protein in several tissues, lack aspartyl ␤-hydroxylase activity in liver preparations, and exhibit no hydroxylation of the epidermal growth factor (EGF) domain of clotting Factor X. In addition to reduced fertility in females, BAH null mice display several developmental defects including syndactyly, facial dysmorphology, and a mild defect in hard palate formation. The developmental defects present in BAH null mice are similar to defects observed in knock-outs and hypomorphs of the Notch ligand Serrate-2. In this work, ␤-hydroxylation of Asp residues in EGF domains is demonstrated for a soluble form of a Notch ligand, human Jagged-1. These results along with recent reports that another post-translational modification of EGF domains in Notch gene family members (glycosylation by Fringe) alters Notch pathway signaling, lends credence to the suggestion that aspartyl ␤-hydroxylation may represent another post-translational modification of EGF domains that can modulate Notch pathway signaling. Previous work has demonstrated increased levels of BAH in certain tumor tissues and a role for BAH in tumorigenesis has been proposed. The role of hydroxylase in tumor formation was tested directly by crossing BAH KO mice with an intestinal tumor model, APCmin mice. Surprisingly, BAH null/APCmin mice show a statistically significant increase in both intestinal polyp size and number when compared with BAH wild-type/APCmin controls. These results suggest that, in contrast to expectations, loss of BAH catalytic activity may promote tumor formation.

Expression of aspartyl beta-hydroxylase and its clinicopathological significance in hepatocellular carcinoma

“…This suggests that in these transmembrane proteins, the extracellular domain regulates signal transduction by the cytoplasmic domain. 12,[24][25][26] In vivo and in vitro experiments have demonstrated that highlevel aspartyl beta-hydroxylase expression is linked to malignant transformation and invasive growth of malignant neoplasms. 27,28 It has been demonstrated that overexpression of the aspartyl beta-hydroxylase cDNAs in NIH-3T3 cells induces a transformed phenotype manifested by increased numbers of transformed foci, anchorage-independent growth, and tumorigenesis in nude mice.…”

N‐glycosylation of CD97 within the EGF domains is crucial for epitope accessibility in normal and malignant cells as well as CD55 ligand binding

“…Aspartyl betahydroxylation and O-fucose glycosylation of EGF domains modulate Notch-receptor signaling. 29,30 Despite the unusually high serine/threonine content, O-glycosylation seems to be less relevant to CD97 molecular weight and interactions. Neither O-glycosidase treatment of immunoprecipitates nor inhibition of O-glycosylation in culture influenced CD97 molecular weight or mAb and CD55 binding.…”

Collagen and calcium-binding EGF domains 1 is frequently inactivated in ovarian cancer by aberrant promoter hypermethylation and modulates cell migration and survival

“…Female BAH knockout mice have developmental abnormalities and decreased fertility, and are more susceptible to tumour formation (Dinchuk et al, 2002). If CCBE1 activity is modulated by hydroxylation of Asp/Asn residues by BAH, it could be predicted that silencing of CCBE1 may result in a similar tumourpromoting effect to that of BAH loss, although this remains to be determined.…”