Identification of a Missense Mutation (G329A; Arg110→ Gln) in the Human FUT7 Gene

Abstract

The human FUT7 gene codes for the ␣1,3-fucosyltransferase VII (Fuc-TVII), which is involved in the biosynthesis of the sialyl Lewis x (SLe x ) epitope on human leukocytes. The FUT7 gene has so far been considered to be monomorphic. Neutrophils isolated from patients with ulcerative colitis were examined for apparent alterations in protein glycosylation patterns by Western blot analysis using monoclonal antibodies directed against SLe x and SLe x -related epitopes. One individual showed lower levels of SLe x expression and an elevated expression of CD65s compared to controls. The coding regions of the FUT7 gene from this individual were cloned, and a G329A point mutation (Arg 110 3 Gln) was found in one allele, whereas the other FUT7 allele was wild type. No Fuc-TVII enzyme activity was detected in COS-7 cells transiently transfected with the mutated FUT7 construct. The FUT7 Arg 110 is conserved in all previously cloned vertebrate ␣1,3-fucosyltransferases. Polymerase chain reaction followed by restriction enzyme cleavage was used to screen 364 unselected Caucasians for the G329A mutation, and a frequency of <1% for this mutation was found (3 heterozygotes). Genetic characterization of the family members of one of the additional heterozygotes identified one individual carrying the G329A mutation in both FUT7 alleles. Peripheral blood neutrophils of this homozygously mutated individual showed a lowered expression of SLe x and an elevated expression of CD65s when analyzed by Western blot and flow cytometry. The homozygous individual was diagnosed with ulcer disease, non-insulin-dependent diabetes, osteoporosis, spondyloarthrosis, and Sjö gren's syndrome but had no history of recurrent bacterial infections or leukocytosis.

Characterization of EBV‐transformed B‐cells Established from an Individual Homozygously Mutated (G329A) in the FUT7α1,3‐fucosyltransferase Gene

“…The level of FUT7 mRNA was similar in IWO and SIGN cells, indicating equal levels of FUT7 transcription. Western blot, using a polyclonal anti-serum directed against Fuc-TVII, detected a strongly stained band at 55 kDa in IWO cell lysates, which is similar to what was found in COS-7 cells transfected with FUT7 and presumably represents the fully processed form of Fuc-TVII [16]. This band was much weaker when SIGN cells were analysed, indicating a possible effect of the G329A mutation on enzyme stability in these cells.…”

“…IWO cells showed weak staining of a 39-kDa band and strong staining of a 55-kDa band. These two bands, possibly representing unprocessed and fully glycosylated variants of the Fuc-TVII enzyme, respectively, were also seen in Western blot analysis of COS-7 cells transfected with a pSI-FUT7wt plasmid using the same polyclonal antibody [16]. SIGN cells showed the same bands, but the expression of the 55-kDa band was considerably weaker.…”

“…These mice showed severely reduced selectin-dependent binding of leukocytes [15]. We have previously identified an individual that was homozygous for a G329A missense mutation in the FUT7 gene [16]. Granulocytes from this individual showed decreased expression of SLe x but still interacted with both P-and E-selectin under flow conditions [17].…”

“…The assay was performed as previously described [16]. Washed IWO and SIGN cells were lysed and denatured, and the extracts were analysed by denaturating SDS-PAGE.…”

“…We previously reported the presence of a missense mutation (G329A) in the human gene encoding for Fuc-TVII, FUT7 [16]. The mutation results in an amino acid shift of Arg to Gln at position 110 of the protein.…”