Secreted and membrane attractin result from alternative splicing of the human ATRN gene

Abstract

Attractin, initially identified as a soluble human plasma protein with dipeptidyl peptidase IV activity that is expressed and released by activated T lymphocytes, also has been identified as the product of the murine mahogany gene with connections to control of pigmentation and energy metabolism. The mahogany product, however, is a transmembrane protein, raising the possibility of a human membrane attractin in addition to the secreted form. The genomic structure of human attractin reveals that soluble attractin arises from transcription of 25 sequential exons on human chromosome 20p13, where the 3' terminal exon contains sequence from a long interspersed nuclear element-1 (LINE-1) retrotransposon element that includes a stop codon and a polyadenylation signal. The mRNA isoform for membrane attractin splices over the LINE-1 exon and includes five exons encoding transmembrane and cytoplasmic domains with organization and coding potential almost identical to that of the mouse gene. The relative abundance of soluble and transmembrane isoforms measured by reverse transcription-PCR is differentially regulated in lymphoid tissues. Because activation of peripheral blood leukocytes with phytohemagglutinin induces strong expression of cell surface attractin followed by release of soluble attractin, these results suggest that a genomic event unique to mammals, LINE-1 insertion, has provided an evolutionary mechanism for regulating cell interactions during an inflammatory reaction.

Figure 1

(A) The sequence represented by AB011120 has been mapped to 20p13 (8), falling between the sequence-tagged site (STS) markers D20S113 and D20S97 that define a region of approximately 3 Mb at a distance of 6–9 Mb from the telomere. Known genes mapped to this region include small nuclear ribonucleoprotein polypeptide B/B1 (SNRPB), transglutaminase 3 (TGM3), and cell division cycle 25B phosphatase (CDC25hu2). The BAC clones identified as containing attractin exons map within a 1-Mb region 8–9 Mb from the telomere (white bar). (B) Representation of the exons coding for the 3′ ends of soluble attractin (exons 22–25) and membrane attractin (exons 22–24 and 26–30). Exon size is presented in base pairs; numbers in parentheses represent size of noncoding sequence. Intron size (kb) was determined by PCR amplification, and in some instances, confirmed by direct genomic sequencing. The thin shaded bars below represent the PCR-amplified regions used to determine relative expression of soluble and membrane mRNA, and the numbers above each bar represent the size of the amplified fragments.

Figure 2

Purified serum attractin, recombinant soluble attractin, and membrane attractin (lysates of transiently transfected 293T cells) were separated by 7.5% SDS/PAGE and detected with affinity-purified rabbit anti-attractin (Left) or anti-C-terminal myc tag (Right) by Western blotting and chemiluminescent detection of bound antibody.

Figure 3

Comparison of exon organization of human membrane attractin and C. elegans F33C8.1. Membrane attractin does not use exon 25. Similarity was determined by using the clustal-x alignment algorithm with default settings (34). Similarity (%) of each exon with the corresponding amino acid sequence in the related genomic sequence is depicted on the vertical scale, where greater similarity results in greater proximity. The shaded boxes represent identified domains of high similarity shared by attractin and F33C8.1 (TM, transmembrane domain).

Figure 4

Promoter sites (in bold) were detected by using the matinspector package (100% identity for core region, 90% identity with consensus flanking residues). The capitalized residues within putative sites represent the core binding motifs. * indicate in-frame stop codons; “+1“ marks the putative start codon and numbering is relative to this. The underline indicates the longest 5′ UTR identified so far in screening cDNA libraries.

Figure 5

First-strand cDNA immune tissue [primed from the poly(A) tail] that had been normalized for four housekeeping genes was tested for representation of soluble and membrane attractin. The lower panels depict amplification of a G3PDH (1 kb) fragment for all of the samples tested. Soluble attractin (412 bp) was amplified by using the primers depicted in Fig. 1B. Membrane attractin (668 bp) was amplified by using primers within the 3′ UTR. Identical results were obtained by using the primers designed from coding sequence (552 bp) but required five additional cycles to obtain a strong signal. This is a result of first-strand priming from poly(A) and the length of the membrane attractin 3′ UTR (>4 kb) leading to under-representation of the coding sequence.