We previously reported the cloning of an intestinal zinc transporter hZTL1 which, when expressed in human intestinal Caco-2 cells with a C-terminal Myc epitope tag, was located at the apical membrane (Cragg et al. 2002). Here, we confirm the localization of hZTL1 in Caco-2 cells and report the location of hZTL1 in sections of human jejunum using an hZTL1 antipeptide antibody.

Rabbit anti-hZTL1 antibody was raised against a synthetic peptide corresponding to amino acids 190-211 of hZTL1 (ILSSPSKRGQKGTLIGYSPEGT) and was affinity purified. Human jejunum was obtained from cadaver donors with appropriate ethics committee approval. Frozen sections (7 µm) were fixed for 30 min on ice with 4 % (w/v) paraformaldehyde in PBS. Caco-2 cells, grown for 14 days on polycarbonate filters, were fixed with 100 % methanol for 5 min at room temperature. Sections or cells were permeabilized in 0.1 % (v/v) Triton X-100 in PBS and incubated in blocking solution (5 % w/v BSA, 10 % (v/v) goat serum, 0.1 % Triton X-100 in PBS). After incubation overnight at 4°C with anti-hZTL1 antibody (1:100), slides were washed in PBS and samples were then incubated with FITC-conjugated goat anti-rabbit IgG (1:500) for 1 h at room temperature. Caco-2 cells were subsequently treated with propidium iodide to reveal nuclear staining or with a chromogenic alkaline phosphatase stain. Sections or filters were mounted in fluorescence mounting medium under a sealed coverslip and visualized using a Leica confocal microscope.

Specific FITC staining was localized at the apical membrane of human jejunum and was concentrated towards the villus tip. In Caco-2 cells, staining was observed exclusively at the apical membrane and co-localized with alkaline phosphatase activity. These data are consistent with the established location of hZTL1 in Caco-2 cells using the Myc-tagged construct (Cragg et al. 2002).

Comparison of the cDNA sequences of hZTL1 and ZnT5 (Kambe et al. 2002) reveals that they are splice variants of the same gene. The antibody used in the study reported here corresponds to a region common to both hZTL1 and ZnT5. We have previously reported that the ZnT5 variant, shown to localize to intracellular vesicles in pancreas (Kambe et al. 2002), is not detected in Caco-2 cells by RT-PCR (Russi et al. 2003). The absence of a vesicular staining pattern in either human intestine or Caco-2 cells reported here is consistent with the expression of only hZTL1 in intestine and adds to the evidence that hZTL1/ZnT5 splice variants are expressed in a tissue-specific manner.

This work was supported by BBSRC grant 13D/11012