Alternatively spliced form of human thyroperoxidase, TPOzanelli: activity, intracellular trafficking, and role in hormonogenesis

Thyroperoxidase (TPO), a type I transmembrane heme containing glycoprotein, catalyzes iodide organification and thyroid hormone synthesis. One of the two main alternatively spliced forms of this enzyme, TPOzanelli, which is present in Graves's disease thyroid tissue, has a cytoplasmic domain completely modified. In the first stage of this study, the results of RT-PCR experiments showed that the TPOzanelli mRNA is present in normal thyroid tissue. We then generated CHO cell lines expressing the wild-type TPO (TPO1) and the alternatively spliced form TPOzanelli. Upon investigating a panel of 12 mAbs directed against the extracellular domain of TPO1 and sera from patients with a high titer of TPO autoantibodies, we observed that (i) the three-dimensional structure of this domain is similar in both isoforms; (ii) the autoantibodies recognize TPOzanelli as well as TPO1. The results of pulse chase and cell surface biotinylation experiments showed that the TPOzanelli has a shorter half-life (7 versus 11 h) and is expressed at the cell surface in lesser amounts than TPO1 (7 versus 15%). The total enzymatic activity and cell surface activity were determined in CHO cells expressing TPO1 and TPOzanelli, and TPO1 and TPOzanelli were found to have similar levels of activity. It was established that approximately 20% of the TPO purified from a Graves' disease thyroid gland was precipitated by polyclonal antibodies directed against a specific part of the cytoplasmic tail of TPOzanelli. This confirmed that the protein corresponding to the mRNA is present in the thyroid tissue. All in all, these results indicate that TPOzanelli can be expected to play a role in thyroid hormone synthesis and in thyroid autoimmunity.     

Novel exons located more than 200 kb downstream of the previously described 3' exon of the K-sam gene for generating activated forms of KGF receptor

The K-sam gene was first identified as an amplified gene in the poorly differentiated types, especially in the scirrhous type, of gastric cancers. We have recently found and reported that the carboxyl-terminal exons of K-sam are frequently deleted in the scirrhous type of gastric cancer. The deletion generates preferential expression of at least six novel K-sam-II mRNAs: K-sam-IIH1, -IIH2 and -IIH3/O4, and K-sam-IIO1, -IIO2, and -IIO3, which encode novel proteins lacking the transformation-inhibitory sequence or activated K-sam proteins. In this study, we investigated expression of the previously described K-sam-IIC1 and -IIC3 mRNAs and the novel six K-sam-II mRNAs in 14 gastric cancer cell lines, 7 breast cancer cell lines, and 20 human normal tissues. All the six novel K-sam-II mRNAs were expressed preferentially in the cell lines derived from the scirrhous type of gastric cancers but not in the 7 breast cancer cell lines and the 20 human normal tissues. We further determined the positional relationship of four exons of H1, O1, O2, and O3 out of the six exons of H1, H2, H3/O4, O1, O2, and O3, and found that these four novel K-sam exons were located more than 200 kb downstream of the previously described carboxyl-terminal exon of the K-sam gene. Expression of K-sam-IIH1, -IIO1, and -IIO2 mRNAs encoding activated K-sam products in the scirrhous type of gastric cancer cell lines HSC39, OCUM2M, HSC59, and HSC60 was not due to the deletion of the C1 exon of K-sam.     

Generation of a biologically active, secreted form of human thyroid peroxidase by site-directed mutagenesis

Using site-directed mutagenesis, we introduced two stop codons immediately upstream of the putative transmembrane domain in human thyroid peroxidase (hTPO) cDNA, truncating the carboxyl terminus of hTPO (933 amino acids) by 85 residues. Mutated hTPO cDNA, inserted into a eukaryotic expression vector, was stably transfected into Chinese hamster ovary (CHO) cells. Immunoprecipitation of cellular 35S-methionine-labeled proteins with Hashimoto's serum revealed a 105-101 kilodalton doublet. In contrast, cells transfected with wild-type hTPO yielded a 112-105 kilodalton doublet. In pulse-chase experiments, CHO cells expressing the truncated hTPO protein secreted immunoprecipitable TPO into the culture medium after 4 h of chase, with levels accumulating progressively over a 24-h period. In contrast, CHO cells expressing wild-type hTPO released no immunoprecipitable TPO into the culture medium. The secreted, truncated form of hTPO appeared as a single band of lesser electrophoretic mobility, as opposed to the doublet expressed within cells. TPO enzymatic activity was present in conditioned media from CHO cells transfected with the mutated hTPO, but was absent in media from cells expressing wild-type hTPO. The stability of the mutated protein appeared similar to that of wild-type hTPO. In summary, we have generated a mutated, secreted form of hTPO that is enzymatically active and immunologically intact. Our data confirm the existence of a transmembrane domain in hTPO, and that hTPO is predominantly an enzyme with an extracellular orientation. The secreted form of hTPO has the potential for generating large amounts of soluble TPO protein for use in future structural and immunological studies.     

Differential expression of CD45 isoforms is controlled by the combined activity of basal and inducible splicing-regulatory elements in each of the variable exons

The human CD45 gene encodes five isoforms of a transmembrane tyrosine phosphatase that differ in their extracellular domains as a result of alternative splicing of exons 4-6. Expression of the CD45 isoforms is tightly regulated in peripheral T cells such that resting cells predominantly express the larger CD45 isoforms, encoded by mRNAs containing two or three variable exons. In contrast, activated T cells express CD45 isoforms encoded by mRNAs lacking most or all of the variable exons. We have previously identified the sequences within CD45 variable exon 4 that control its level of inclusion into spliced mRNAs. Here we map the splicingregulatory sequences within CD45 variable exons 5 and 6. We show that, like exon 4, exons 5 and 6 each contain an exonic splicing silencer (ESS) and an exonic splicing enhancer (ESE), which together determine the level of exon inclusion in na?ve cells. We further demonstrate that the primary activation-responsive silencing motif in exons 5 and 6 is homologous to that in exon 4 and, as in exon 4, binds specifically to the protein heterogeneous nuclear ribonucleoprotein L. Together these studies reveal common themes in the regulation of the CD45 variable exons and provide a mechanistic explanation for the observed physiological expression of CD45 isoforms.     

Cloning and expression of novel isoforms of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from bovine heart

Distinct 6-phosphofructo-2-kinase (PFK-2)/fructose 2,6-bisphosphatase (FBPase-2) cDNAs were cloned from bovine heart, showing that PFK-2/FBPase-2 gene B, which contains 16 exons, codes for at least five mRNAs. Three of them (B1, B2, B4) could encode the 58,000-M_r isozyme. In B2 mRNA, exon 15 encodes four more residues than in Bl. In B4 mRNA, exon 15 encodes six more residues than in B1, butexon 16 (20 residues) is missing. B3 mRNA corresponds to the 54,000-M_r isozyme. It lacks exon 15 and also differs from the other mRNAs in the 5' noncoding region. B5 mRNA encodes a truncated form. When expressed in E. coli, the recombinant isoforms corresponding to all these mRNAs except B5 exhibited PFK-2 activity.