The isolation and characterization of complementary DNAs (cDNAs) which code for an epithelial antigen aberrantly expressed in human breast tumor tissue are described here. The only information regarding the primary structure of this potentially important antigen has been a 20-amino-acid repeat motif. We now report the complete amino acid sequences of different forms of the human epithelial tumor antigen as deduced from the nucleotide sequence of isolated non-repeat cDNAs. The diversity of protein forms is generated by a series of alternative splicing events that occur in the regions located upstream and downstream to a central tandem repeat array. Isolated cDNAs coding for the upstream region show that differential usage of alternative splice acceptor sites may generate two protein forms containing putative signal peptides of varying hydrophobicities. The complexity of possible antigen forms is further compounded by alternative splicing events occurring in the region 3' to the repeat array. The isolated cDNAs 3' to the tandem repeats indicate that whereas one mRNA transcript is colinear with the gene, and defines an open reading frame (ORF) containing 160 amino acids downstream to the repeat array, a second cDNA correlates with a mRNA that is generated by a series of splicing events. The deduced amino acid sequence of the spliced cDNA contains an ORF that is identical for 149 amino acids downstream to the repeat array with the amino acid sequence of the unspliced cDNA. At this point it diverges and continues for an additional 179 amino acids. The sequence contains a highly hydrophobic 28-amino-acid peptide, located towards the carboxyl terminus, that may correspond to a transmembrane region. The cDNAs and deduced amino acid sequences, presented here, define the complete amino acid sequences of the epithelial tumor antigen and demonstrate the existence of multiple protein forms that probably localize to different cellular and extracellular compartments.
The collagen family of proteins consists of 19 types encoded by 33 genes. One of the more recently discovered collagens is the alpha1 chain of type XV. Type XV collagen is comprised of a 577-amino-acid, highly interrupted, triple-helical region that is flanked by amino and carboxy noncollagenous domains of 555 and 256 residues, respectively. To address questions of where this collagen is localized and what its function may entail, we produced a bacteria-expressed recombinant protein representing the first half of the type XV collagen carboxy-terminal domain in order to generate highly specific polyclonal antisera. Immunoscreening of an expression library with the affinity-purified antibody revealed three clones coding for part of the type XV triple-helical region and the entire noncollagenous carboxy-terminus. Western blot analysis of human tissue homogenates identified a 116-kDa collagenase-sensitive protein and a 27-kDa collagenase-resistant fragment, whose electrophoretic mobilities were unchanged in the presence and absence of reductant. Northern blot hybridization to human tissue RNAs indicated that type XV has a prevalent and widespread distribution. To determine the precise localization of type XV collagen, immunohistochemical analyses at the light- and electron-microscopic levels were performed. Type XV exhibited a surprisingly restricted and uniform presence in many human tissues as evidenced by a strong association with vascular, neuronal, mesenchymal, and some epithelial basement membrane zones. These data suggest that type XV collagen may function in some manner to adhere basement membrane to the underlying connective tissue stroma.
A previously unknown collagen cDNA clone, PF19, was isolated from a human placenta library. The 2.1-kilobase Insert has a complete open reading frame of709 amIno adds that includes 12 amino acids ofthe NHrterminal domain, a principally collagenous region of 577 residues, and 120 residues of the noncollagenous COOH terminus. The cIanous part of the sequence encoded by PF19 Is characterized by 13 interruptions ranging in size from 2 to 45 amino aids. Within four interruptions are consensus sequences for attachment of serine-linked glycosamlnoglycans and araginelinked oligosaccharides suggeting that this collagen may be extensively glycosylated. A synthetic decapeptide-reprting a sequence at the b nng of the COOH-terminal ncollagenous domain was used to prepare an antibody in rabbits. This antiserum deteced a 125-kDa bacteria lagenase-senstive protein in Western blots ofHeLa cell lysate. Consistent with the size of the collagen chin, Northern blot hybridization revealed a major transcript of 5.3 kilobases and two minor ones of 4.7 and 4.4 kilobases that are present in cultured human fibroblasts but absent from umbilcal vein endothelial celis. We propose that the previously unidentified polypeptide descibed in this report be designated the al chain of type XV collagen.
Objective. Since nucleus pulposus cells reside under conditions of hypoxia, we determined if the expression of ANK, a pyrophosphate transporter, is regulated by the hypoxia-inducible factor (HIF) proteins.Methods. Quantitative reverse transcriptionpolymerase chain reaction and Western blot analyses were used to measure ANK expression in nucleus pulposus cells from rats and humans. Transfections were performed to determine the effect of HIF-1/2 on ANK promoter activity.Results. ANK was expressed in embryonic and mature rat discs. Oxygen-dependent changes in ANK expression in nucleus pulposus cells were minimal. However, silencing of HIF-1␣ and HIF-2␣ resulted in increased ANK expression and up-regulation of promoter activity. HIF-mediated suppression of ANK was validated by measuring promoter activity in HIF-1-null embryonic fibroblasts. Under conditions of hypoxia, there was induction of promoter activity in the null cells as compared with the wild-type cells. Overexpression of HIF-1␣ and HIF-2␣ in nucleus pulposus cells resulted in a significant suppression of ANK promoter activity. Since the ANK promoter contains 2 hypoxiaresponsive elements (HREs), we performed site-directed mutagenesis and measured promoter activity. We found that HIF-1 can bind to either of the HREs and can suppress promoter activity; in contrast, HIF-2 was required to bind to both HREs in order to suppress activity. Finally, analysis of human nucleus pulposus tissue showed that while ANK was expressed in normal tissue, there was increased expression of ANK along with alkaline phosphatase in the degenerated state.Conclusion. Both HIF-1 and HIF-2 serve as negative regulators of ANK expression in the disc. We propose that baseline expression of ANK in the disc serves to prevent mineral formation under physiologic conditions.Within the spine, the vertebrae are separated by a complex tissue, the intervertebral disc. At the periphery of the disc, a ligamentous tissue, the anulus fibrosus, encloses the proteoglycan-rich nucleus pulposus. Although details of the ontology of cells of the adult nucleus pulposus are obscure, it is known that they are derived from the notochord (1), an embryonic tissue with a limited blood supply. The superior and inferior boundaries of the intervertebral disc are formed by the cartilage end plates. A limited number of blood vessels infiltrate the end plates and the outer anulus fibrosus but do not enter the nucleus pulposus (2,3). For this reason, there is considerable support for the view that the nucleus pulposus cells reside in a hypoxic environment (4)(5)(6). Surprisingly, while the disc contains both fibrous proteins and a hydrated extracellular matrix, calcified deposits are absent in the nucleus pulposus and the anulus fibrosus in healthy state.
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