Hiroshi Nakazato

Primary structure of human carcinoembryonic antigen (CEA) deduced from cDNA sequence.

The cDNAs corresponding to the mRNA encoding a polypeptide which is immunoreactive with the antisera specific to carcinoembryonic antigen (CEA) (1) are cloned. The amino acid sequences deduced from the nucleotide sequences of the cDNAs show that it is synthesized as a precursor with a signal peptide followed by 668 amino acids of the putative mature CEA peptide, whose N-terminal 24 amino acids and amino acids 286 to 295 exactly coincide with those known for N-terminal sequences of CEA (2) and NFA-1 (3), respectively. The first 108 N-terminal residues are followed by three very homologous repetitive domains of 178 residues each and then by 26 mostly hydrophobic residues which probably comprise a membrane anchor. Each repetitive domains contains 4 cysteines at precisely the same positions and as many as 28 possible N-glycosylation sites are found in the CEA peptide region agreeing with high carbohydrate content of purified CEA.

Identification of human placental leucine aminopeptidase as oxytocinase

Human placental leucine aminopeptidase (P-LAP) was purified from retroplacental serum for the first time by serial chromatography on columns of Matrex Blue A, DEAE-Sepharose CL-6B, phenyl-Sepharose 4B, chelating-Sepharose, and Sepharose CL-6B. The purified P-LAP was apparently homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the apparent molecular weight (Mr) was estimated to be 210,000. By comparing P-LAP activity with cystine aminopeptidase activity, we concluded that both activities were shared by the same molecule. We also examined the hydrolytic activity of P-LAP using naturally occurring peptide hormones and found that the enzyme hydrolyzed oxytocin, vasopressin, and angiotensin III. These results suggest that P-LAP shows oxytocinase activity and plays an important role in the regulation of the plasma level of these hormones during pregnancy.

Structure of dog and rabbit precursors of atrial natriuretic polypeptides deduced from nucleotide sequence of cloned cDNA

The structure of precursors of dog and rabbit atrial natriuretic polypeptides was determined by nucleotide sequence analysis of cloned cDNA of mRNA encoding the peptides. The dog and rabbit precursors are 149 and 153 residues long having 23- and 25-residue putative signal peptides at their N-termini respectively. The 28-residue peptide with identical sequence to that of human, which has potent natriuretic activity, is located at the C-terminus of the dog precursor. The 28-residue peptide of identical sequence to that of mouse/rat is located at C-terminus of rabbit precursor followed by additional Arg-Arg sequence which is also found in rat/mouse precursors and is apparently removed during processing.

The carcinoembryonic antigen (CEA) contains multiple immunoglobulin-like domains

The amino acid sequences of human carcinoembryonic antigen deduced from the cDNA sequences have been analysed. This antigen contains seven extracellular domains (previously recognized three highly repetitive domains are further divided into A and B subdomains each) which are strikingly homologous to each other and to immunoglobulin variable regions, poly-Ig receptor and Thy 1.1. The N-terminal domain lacks immunoglobulin-like fold but the other six domains have, suggesting that the CEA belongs to immunoglobulin superfamily.

Polyadenylic Acid Sequences in the Virion RNA of Poliovirus and Eastern Equine Encephalitis Virus

Poliovirus virion RNA contains a single covalently bound sequence of polyadenylic acid which is approximately 49 nucleotides long. A single, slightly longer polyadenylic acid sequence is contained in Eastern Equine Encephalitis virus RNA. Since these viruses are otherwise dissimilar these sequences may play a common role in viral replication, possibly in translation of the viral RNA.

Homotypic and heterotypic Ca++-independent cell adhesion activities of biliary glycoprotein, a member of carcinoembryonic antigen family, expressed on CHO cell surface

Homotypic and heterotypic cell adhesion activities of a carcinoembryonic antigen (CEA) family member, biliary glycoprotein a (BGPa), have been examined. CHO cells transfected with the cDNA for BGPa, CEA, non-specific cross-reacting antigen (NCA) and CGM6 have been used. The BGPa producers showed both homotypic and heterotypic adhesion to CEA and NCA producers. However, they hardly adhered to CGM6 producers. Calcium ion was not required for BGPa-mediated homotypic and heterotypic cell adhesion as well as for the adhesions of other members of CEA family. The results strongly suggested that BGPa may play some important roles through Ca(++)-independent cell adhesion activities.

Production and characterization of monoclonal antibodies to human tumor necrosis factor.

Five stable hybridoma cell lines secreting antibodies to recombinant human tumor necrosis factor (TNF) were established. All monoclonal antibodies belong to the IgG1 subclass. One monoclonal antibody (MAB) (designated as 3B10) neutralizes the L929 cytotoxic activity of natural and recombinant human TNF. Other MABs bind to human TNF but do not neutralize cytotoxic activity. A sandwich enzyme immunoassay specific to human TNF molecule has been developed using 3B10. This assay measures only biologically active human TNF molecules and is as sensitive as a bioassay which measures the cytotoxic activity on L929 cells. These results show that 3B10 is quite useful in studying the biological functions of TNF.

Placental leucine aminopeptidase/oxytocinase in maternal serum and placenta during normal pregnancy

Placental leucine aminopeptidase (P-LAP), which is identical with cystine aminopeptidase as oxytocinase, was found to be homologous with rat insulin-regulated membrane aminopeptidase (IRAP) by sequence comparison. In the current study, we determined the P-LAP levels in maternal serum and placenta during healthy pregnancy. P-LAP activities in maternal serum increased with gestation and rose to the peak of 80 IU/ml at 38 weeks of gestation. Northern blot analysis revealed the increase of P-LAP mRNA levels in placenta in the third trimester compared to the first trimester. P-LAP protein and related activities could be detected in the conditioned medium of placental tissue, while they could not be detected in that of human umbilical vein endothelial cells. Immunohistochemically P-LAP was positively stained in the apical membrane of syncytiotrophoblast cells throughout the gestation. These results established the normal range of serum and tissue P-LAP levels during pregnancy and the possible source of serum P-LAP, which will be helpful to elucidate the physiological and clinical roles of P-LAP/oxytocinase/IRAP.

Metastatic potential of human colorectal carcinoma SW1222 cells transfected with cDNA encoding carcinoembryonic antigen

In order to examine a role of carcinoembryonic antigen (CEA) in metastasis, cDNA encoding CEA was introduced into a clone of human colorectal carcinoma SW1222 cells. Western blot analysis revealed that all transfectants express CEA of 180 kDa while the parent clone does not. In the transfectants, the level of CEA expression in clone 3 was higher than that of clone 1. Clone 3 formed aggregates rapidly after suspended by trypsinization while clone 1 did not. In experimental metastasis assay where tumor cells were injected intrasplenically, clone 3 exhibited a higher liver-metastatic activity than clone 1. Fab fragment of anti-CEA antibody significantly inhibited both the cell aggregation and the liver metastases caused by clone 3. These findings suggested that CEA expressed on the cell surface may play an important role in hepatic metastasis from colorectal carcinoma, possibly through its cell adhesion activity.

A single amino acid substitution converts cytochrome P45014DM to an inactive form, cytochrome P450SG1: Complete primary structures deduced from cloned DNAs

Genes for lanosterol 14-demethylase, cytochrome P450(14DM), and a mutated inactive cytochrome P450SG1 were cloned from S. cerevisiae strains D587 and SG1, respectively. A single nucleotide change resulting in substitution of Asp for Gly-310 of cytochrome P450(14DM) was found to have occurred in cytochrome P450SG1. In this protein the 6th ligand to heme iron is a histidine residue instead of a water molecule, which may be the ligand for the active cytochrome P450(14DM). Molecular models of the active sites of the cytochrome P450(14DM) and cytochrome P450SG1 were built by computer modeling on the basis of the known structure of that of cytochrome P450CAM whose crystallographic data are available. The mechanisms which may cause a histidine residue to gain access to the heme iron are discussed.