Structure and Expression of Rat Osteosarcoma (ROS 17/2.8) Alkaline Phosphatase: Product of a Single Copy Gene

• Published in: Proceedings of the National Academy of Sciences - PNAS 1988, Vol.85 (2), p.319-323
• Main Authors: Thiede, Mark A., Yoon, Kyonggeun, Golub, Ellis E., Noda, Masaki, Rodan, Gideon A.
• Format: Article
• Language: English
• Subjects: Alkaline Phosphatase - genetics; Amino Acid Sequence; Amino acids; Animals; Base Sequence; Biological and medical sciences; Cell Line; Chemical bases; CHO cells; Complementary DNA; DNA; DNA probes; Enzymes; Fundamental and applied biological sciences. Psychology; Gene expression; Genes; Messenger RNA; Models, Molecular; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Osteosarcoma - enzymology; Osteosarcoma - genetics; Protein Conformation; Rats; RNA; Sarcoma, Experimental - enzymology; Sarcoma, Experimental - genetics; Transcription, Genetic
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.85.2.319

Alkaline phosphatase [ALP; orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] is a ubiquitous enzyme of unknown function expressed at high levels in cells of mineralizing tissues. To study the structure, function, and expression of ALP, a full-length cDNA of rat ALP (2415 bases) was isolated from a ROS 17/2.8 osteosarcoma cell λ gt10 cDNA library. The predicted amino acid sequence spans 524 residues and includes an N-terminal signal peptide of 17 amino acids, the phosphohydrolase active site, a rather hydrophilic backbone with five potential N-glycosylation sites, and a short hydrophobic C-terminal sequence. ALP negative CHO cells transfected with an expression vector containing the ALP coding sequences express ALP. The rat bone, liver, and kidney ALP shows remarkable 90% homology with the corresponding human enzyme, the most divergent region being the C-terminal hydrophobic domain through which the enzyme may be anchored to the plasma membrane. The rat ALP also shows 50% homology with the human placental and intestinal ALP and 25% homology with the Escherichia coli ALP. The amino acids involved in catalysis show nearly complete homology among all known ALP sequences, suggesting that these enzymes evolved from a common ancestral gene. The rat ALP cDNA pRAP 54, used as a hybridization probe in RNA blot analysis of several tissues that express ALP, revealed the presence of an ALP mRNA of ≈ 2500 bases. Furthermore, hybridization patterns derived from Southern blot analysis of rat chromosomal DNA offered molecular evidence that the ALP expressed in ROS 17/2.8 osteosarcoma and various rat tissues, excluding the intestine, is the product of the same single copy gene.