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Cellular expression, trafficking, and function of two isoforms of human ULBP5/RAET1G.

Eagle RA, Flack G, Warford A, Martínez-Borra J, Jafferji I, Traherne JA, Ohashi M, Boyle LH, Barrow AD, Caillat-Zucman S, Young NT, Trowsdale J - PLoS ONE (2009)

Bottom Line: Both endogenously expressed and transfected RAET1G1 was mainly found inside the cell, with a minority of the protein reaching the cell surface.We demonstrate that the expression patterns of ULBP5RAET1G are very similar to the well-characterised NKG2D ligand, MICA.However the two isoforms of ULBP5/RAET1G have very different cellular localisations that are likely to reflect unique functionality.

View Article: PubMed Central - PubMed

Affiliation: Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Addenbrookes Hospital, Cambridge, UK. robeagle@caltech.edu

ABSTRACT

Background: The activating immunoreceptor NKG2D is expressed on Natural Killer (NK) cells and subsets of T cells. NKG2D contributes to anti-tumour and anti-viral immune responses in vitro and in vivo. The ligands for NKG2D in humans are diverse proteins of the MIC and ULBP/RAET families that are upregulated on the surface of virally infected cells and tumours. Two splicing variants of ULBP5/RAET1G have been cloned previously, but not extensively characterised.

Methodology/principal findings: We pursue a number of approaches to characterise the expression, trafficking, and function of the two isoforms of ULBP5/RAET1G. We show that both transcripts are frequently expressed in cell lines derived from epithelial cancers, and in primary breast cancers. The full-length transcript, RAET1G1, is predicted to encode a molecule with transmembrane and cytoplasmic domains that are unique amongst NKG2D ligands. Using specific anti-RAET1G1 antiserum to stain tissue microarrays we show that RAET1G1 expression is highly restricted in normal tissues. RAET1G1 was expressed at a low level in normal gastrointestinal epithelial cells in a similar pattern to MICA. Both RAET1G1 and MICA showed increased expression in the gut of patients with celiac disease. In contrast to healthy tissues the RAET1G1 antiserum stained a wide variety or different primary tumour sections. Both endogenously expressed and transfected RAET1G1 was mainly found inside the cell, with a minority of the protein reaching the cell surface. Conversely the truncated splicing variant of RAET1G2 was shown to encode a soluble molecule that could be secreted from cells. Secreted RAET1G2 was shown to downregulate NKG2D receptor expression on NK cells and hence may represent a novel tumour immune evasion strategy.

Conclusions/significance: We demonstrate that the expression patterns of ULBP5RAET1G are very similar to the well-characterised NKG2D ligand, MICA. However the two isoforms of ULBP5/RAET1G have very different cellular localisations that are likely to reflect unique functionality.

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Immunohistochemical localisation of RAET1G1 in normal human tissues.The anti-RAET1G antiserum was used to stain a tissue array of 344 cores from 172 donors that represented 35 normal tissues. Staining was very infrequently observed, but was present in (A) epithelial cells of colon (B) a minority of tubular cells in kidney, (C) majority of endocrine cells of the anterior pituitary and (D) cells lining thyroid follicles. The pituitary staining may represent true RAET1G1 expression or epitope cross reactivity, as has been shown to occur with other antibodies [53], [54]. All other normal tissues were negative. Pre-immune serum did not stain any of these tissues; (E) colon, (F) kidney, (G) pituitary, and (H) thyroid.
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pone-0004503-g003: Immunohistochemical localisation of RAET1G1 in normal human tissues.The anti-RAET1G antiserum was used to stain a tissue array of 344 cores from 172 donors that represented 35 normal tissues. Staining was very infrequently observed, but was present in (A) epithelial cells of colon (B) a minority of tubular cells in kidney, (C) majority of endocrine cells of the anterior pituitary and (D) cells lining thyroid follicles. The pituitary staining may represent true RAET1G1 expression or epitope cross reactivity, as has been shown to occur with other antibodies [53], [54]. All other normal tissues were negative. Pre-immune serum did not stain any of these tissues; (E) colon, (F) kidney, (G) pituitary, and (H) thyroid.

Mentions: Limited expression of the NKG2D ligand MICA in normal tissues has been previously reported [28], however no systematic study of other NKG2D ligands has been conducted. We used our RAET1G1 specific antiserum to probe a tissue microarray, comprised of 344 cores from 172 donors, representing 35 normal tissues (Table 1). Purified pre-immune rabbit serum did not stain any core. RAET1G1 antibody staining was demonstrated only in four normal tissues (Figure 3). These include gut epithelium, that has previously been shown to express RAET1G1 transcript [14], and stained with the anti-RAET1G1 antiserum in two of five individuals tested. Expression of RAET1G1 in the gut is also noteworthy as MICA has widely been reported to be constitutively expressed in the gut epithelium, and involved in gut pathology [28], [29], [31], [45].


Cellular expression, trafficking, and function of two isoforms of human ULBP5/RAET1G.

Eagle RA, Flack G, Warford A, Martínez-Borra J, Jafferji I, Traherne JA, Ohashi M, Boyle LH, Barrow AD, Caillat-Zucman S, Young NT, Trowsdale J - PLoS ONE (2009)

Immunohistochemical localisation of RAET1G1 in normal human tissues.The anti-RAET1G antiserum was used to stain a tissue array of 344 cores from 172 donors that represented 35 normal tissues. Staining was very infrequently observed, but was present in (A) epithelial cells of colon (B) a minority of tubular cells in kidney, (C) majority of endocrine cells of the anterior pituitary and (D) cells lining thyroid follicles. The pituitary staining may represent true RAET1G1 expression or epitope cross reactivity, as has been shown to occur with other antibodies [53], [54]. All other normal tissues were negative. Pre-immune serum did not stain any of these tissues; (E) colon, (F) kidney, (G) pituitary, and (H) thyroid.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2637608&req=5

pone-0004503-g003: Immunohistochemical localisation of RAET1G1 in normal human tissues.The anti-RAET1G antiserum was used to stain a tissue array of 344 cores from 172 donors that represented 35 normal tissues. Staining was very infrequently observed, but was present in (A) epithelial cells of colon (B) a minority of tubular cells in kidney, (C) majority of endocrine cells of the anterior pituitary and (D) cells lining thyroid follicles. The pituitary staining may represent true RAET1G1 expression or epitope cross reactivity, as has been shown to occur with other antibodies [53], [54]. All other normal tissues were negative. Pre-immune serum did not stain any of these tissues; (E) colon, (F) kidney, (G) pituitary, and (H) thyroid.
Mentions: Limited expression of the NKG2D ligand MICA in normal tissues has been previously reported [28], however no systematic study of other NKG2D ligands has been conducted. We used our RAET1G1 specific antiserum to probe a tissue microarray, comprised of 344 cores from 172 donors, representing 35 normal tissues (Table 1). Purified pre-immune rabbit serum did not stain any core. RAET1G1 antibody staining was demonstrated only in four normal tissues (Figure 3). These include gut epithelium, that has previously been shown to express RAET1G1 transcript [14], and stained with the anti-RAET1G1 antiserum in two of five individuals tested. Expression of RAET1G1 in the gut is also noteworthy as MICA has widely been reported to be constitutively expressed in the gut epithelium, and involved in gut pathology [28], [29], [31], [45].

Bottom Line: Both endogenously expressed and transfected RAET1G1 was mainly found inside the cell, with a minority of the protein reaching the cell surface.We demonstrate that the expression patterns of ULBP5RAET1G are very similar to the well-characterised NKG2D ligand, MICA.However the two isoforms of ULBP5/RAET1G have very different cellular localisations that are likely to reflect unique functionality.

View Article: PubMed Central - PubMed

Affiliation: Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Addenbrookes Hospital, Cambridge, UK. robeagle@caltech.edu

ABSTRACT

Background: The activating immunoreceptor NKG2D is expressed on Natural Killer (NK) cells and subsets of T cells. NKG2D contributes to anti-tumour and anti-viral immune responses in vitro and in vivo. The ligands for NKG2D in humans are diverse proteins of the MIC and ULBP/RAET families that are upregulated on the surface of virally infected cells and tumours. Two splicing variants of ULBP5/RAET1G have been cloned previously, but not extensively characterised.

Methodology/principal findings: We pursue a number of approaches to characterise the expression, trafficking, and function of the two isoforms of ULBP5/RAET1G. We show that both transcripts are frequently expressed in cell lines derived from epithelial cancers, and in primary breast cancers. The full-length transcript, RAET1G1, is predicted to encode a molecule with transmembrane and cytoplasmic domains that are unique amongst NKG2D ligands. Using specific anti-RAET1G1 antiserum to stain tissue microarrays we show that RAET1G1 expression is highly restricted in normal tissues. RAET1G1 was expressed at a low level in normal gastrointestinal epithelial cells in a similar pattern to MICA. Both RAET1G1 and MICA showed increased expression in the gut of patients with celiac disease. In contrast to healthy tissues the RAET1G1 antiserum stained a wide variety or different primary tumour sections. Both endogenously expressed and transfected RAET1G1 was mainly found inside the cell, with a minority of the protein reaching the cell surface. Conversely the truncated splicing variant of RAET1G2 was shown to encode a soluble molecule that could be secreted from cells. Secreted RAET1G2 was shown to downregulate NKG2D receptor expression on NK cells and hence may represent a novel tumour immune evasion strategy.

Conclusions/significance: We demonstrate that the expression patterns of ULBP5RAET1G are very similar to the well-characterised NKG2D ligand, MICA. However the two isoforms of ULBP5/RAET1G have very different cellular localisations that are likely to reflect unique functionality.

Show MeSH
Related in: MedlinePlus