Peters Plus Syndrome Is a New Congenital Disorder of Glycosylation and Involves Defective O-Glycosylation of Thrombospondin Type 1 Repeats

Jan Hofsteenge,Daniel Hess,Raoul C.M. Hennekam,Saskia A. Lesnik Oberstein,Jeremy J. Keusch

doi:10.1074/jbc.m710251200

Abstract

Peters Plus syndrome is an autosomal recessive disorder characterized by anterior eye chamber defects, disproportionate short stature, developmental delay, and cleft lip and/or palate. It is caused by splice site mutations in what was thought to be a beta1,3-galactosyltransferase-like gene (B3GALTL). Recently, we and others found this gene to encode a beta1,3-glucosyltransferase involved in the synthesis of the disaccharide Glc-beta1,3-Fuc-Omicron-that occurs on thrombospondin type 1 repeats of many biologically important proteins. No functional tests have been performed to date on the presumed glycosylation defect in Peters Plus syndrome. We have established a sensitive immunopurification-mass spectrometry method, using multiple reaction monitoring, to analyze Omicron-fucosyl glycans. It was used to compare the reporter protein properdin from Peters Plus patients with that from control heterozygous relatives. In properdin from patients, we could not detect the Glc-beta1,3-Fuc-Omicron-disaccharide, and we only found Fuc-Omicron-at all four Omicron-fucosylation sites. In contrast, properdin from heterozygous relatives and a healthy volunteer carried the Glc-beta1,3-Fuc-Omicron-disaccharide. These data firmly establish Peters Plus syndrome as a new congenital disorder of glycosylation.

Highlights

Transferase-like gene (B3GALTL) that was originally identified by Heinonen et al [4]
The results presented here are the first to provide functional evidence for the type of glycosylation defect caused by biallelic truncating mutations in the gene encoding ␤3Glc-T, and which are likely to lead to Peter Plus syndrome
In patients that were either homozygous for the donor splice site mutation c.660 ϩ 1G3 A, or carried this mutation on the paternal chromosome and a deletion on the maternal one, a complete loss of glucosylation of O-linked fucose in the TSRs of properdin was observed

Summary

EXPERIMENTAL PROCEDURES

Materials—Monoclonal anti-properdin antibody HYB039-06 and goat anti-human properdin were purchased from the Antibody Shop, Gentofte, Denmark. Individuals 4 and 5 are two brothers with Peters Plus syndrome that are compound heterozygous for the c.660 ϩ 1G3 A exon 8 donor splice site mutation in the B3GALTL on the paternal allele and a ϳ1.5-Mb 13q12.3q13.1, B3GALTL including deletion on the maternal allele. Their healthy parents are individuals 2 and 3 and are heterozygous for the respective mutations in their sons. For quantitative comparison of a specific glycoform of peptide T7 between different properdin samples, we injected 0.5 ␮l of the 5-fold diluted tryptic digest (ϳ40 fmol) for LC-MSMS in the multiple reaction monitoring (MRM) mode. The intensity of the latter was used to normalize all other transitions for the amount of analyzed protein

RESULTS

C T7: WSLWSTWAPCSVTCSEGSQLR

DISCUSSION