Complete primary structure of prostatropin, a prostate epithelial cell growth factor

Bovine brain prostatropin is a potent and essential mitogen for prostate epithelial cell growth. The major form of prostatropin contains 154 amino acid residues in a single amino terminally blocked chain corresponding to a molecular weight of 17,400. The amino acid sequence of the 150 carboxy-terminal residues of prostatropin was derived by Edman degradation of overlapping peptides primarily generated by cleavage at lysyl and glutamyl residues. Analysis of the amino-terminal tetradecapeptide by fast atom bombardment mass spectrometry identified the blocking group as an acetyl moiety, and tandem mass spectrometry provided the sequence of the first 12 residues. Prostatropin residues 15-154 contain the sequence of bovine brain polypeptides recently described as acidic fibroblast growth factor and class I heparin-binding growth factor. The sequence of the first 25 residues of prostatropin is acetyl-Ala-(Gly, Glu)-Glu-Thr-Thr-Thr-Phe-Thr-Ala-Leu-Thr-Glu-Lys-Phe-Asn-Leu-Pro-Leu-Gly -Asn-Tyr-Lys-Lys-Pro. Reduced and carboxymethylated prostatropin exhibits mitogenic activity, suggesting that disulfide bonds among cysteine residues 30, 61, and 97 are not functionally essential. These results demonstrate by rigorous structural analysis that the brain-derived polypeptide previously described only as a mesenchymal and neuroectodermal cell mitogen is also an epithelial cell growth factor that may be involved in support of prostate hyperplasia and adenocarcinoma.     

Structural and antigenic studies of an idiotype-bearing murine antibody to the arsonate hapten.

Mice of strain A/J responded to repeated intraperitoneal injection of Limulus hemocyanin derivatized with arsanilic acid by producing large quantities (approximately 5 mg/mL of ascites fluid) of IgG antibodies specific for this hapten. The antibodies possessed a characteristic idiotypic determinant and exhibited restricted heterogeneity as demonstrated by isoelectric focusing and primary N-terminal amino acid sequence analysis of isolated light and heavy polypeptide chains. Both light- and heavy-chain sequences were comparable to those of myeloma proteins in lack of heterogeneity. The N terminus of the light chain exhibited V KI sequence and only one position in the first 30 residues showed more than one amino acid. No variability was observed in the first 10 N-terminal residues of the heavy chain. Rabbit antiserum to the idiotype blocked binding of hapten by the purified antibody. The presence of both light- and heavy-chain antigenic determinants was required for optimal formation of the idiotypic determinant.     

Complete amino acid sequence of a new murine T-cell growth factor P40

A new murine T-cell growth factor, designated P40, which supports growth of helper T-cells in the absence of interleukin-2, interleukin-4 and antigen has been isolated from helper T-cell lines in sufficient quantities (100 micrograms) to permit its complete amino acid sequence determination. This was achieved by a combination of sensitive peptide mapping using microbore reversed-phase high performance liquid chromatography and automated microsequence analysis. Attempts to obtain N-terminal sequence data on P40 were unsuccessful due to N-terminal blockage of the native molecule. The nature of this N-terminal blocking was established using a combination of amino acid analysis, fast-atom-bombardment mass spectrometry and peptide synthesis. The P40 molecule, a single polypeptide chain comprising 126 amino acid residues, is structurally distinct from other known T-cell growth factors. No similarity was revealed when the amino acid sequence of P40 was compared with other proteins whose biochemical structure is known. The protein sequence data reported here predict four N-linked glycosylation sites in the P40 molecule.     

Characterization of the effects of human placental HGF on rat hepatocytes.

Hepatocyte growth factor (HGF) also known as hepatopoietin A (HPTA) (Michalopoulos, FASEB J., 4:176-187, 1990) is a heparin-binding growth factor whose characterization and tissue distribution have been reported elsewhere. This growth factor was recently cloned and its amino acid sequence determined under the name of hepatocyte growth factor (HGF) (Miyazawa et al., Biochem. Biophys. Res. Commun., 163:967-973, 1989; Zarnegar et al., Biochem. Biophys. Res. Commun., 163:1370-1376, 1989; Nakamura et al., Nature, 342:440-443, 1989). Human placenta is one of the tissues that contains significant amounts of HGF. We isolated HGF from human placenta and characterized its biologic effect on rat hepatocytes. Human placenta HGF was isolated in high purity as a single chain molecule. Single chain HGF stimulated DNA synthesis in primary rat hepatocyte cultures in serum-free medium. The maximal effect was seen at 5-10 ng/ml. The maximal response occurred at 25-48 hours after plating of the hepatocytes. Protein synthesis was also stimulated by HGF in primary rat hepatocyte cultures. There were peak responses at 19-24 and 37-42 hours after plating of the hepatocytes. TGF beta 1 inhibited more than 95% of HGF-induced DNA synthesis but only 25% of HGF-induced protein synthesis. HGF interacted in an additive manner with EGF, a well-known hepatocyte mitogen. There was not an additive interaction between HGF and aFGF. Regenerating liver hepatocytes obtained from rats which underwent two-thirds partial hepatectomies (PHX) also responded to HGF in a dose-dependent manner as the hepatocytes from normal liver.     

Amino acid sequence of porcine spleen cathepsin D light chain

The complete amino acid sequence of the light chain of cathepsin D from porcine spleen has been determined. The light chain consists of a single polypeptide chain with 97 amino acid residues. The sequence is: (formula; see text) The molecular weight of the light chain was calculated from this sequence to be 10,548 (without carbohydrates). A single disulfide bond links two half-cystine residues between positions 46 and 53. A cysteine residue is located at position 27. The light chain sequence is extensively homologous to the NH2-terminal sequence of other aspartyl proteases. It shows a 59% identity with the sequence of mouse submaxillary gland renin and a 49% identity with that of porcine pepsin. A single glycosylation site is located at residue 70 of the cathepsin D light chain. This site corresponds to position 67 of pepsin by homology. The active site aspartyl residue, corresponding to Asp-32 of pepsin, is located at residue 33 in the cathepsin D light chain.     

Primary structure of the silk fibroin light chain determined by cDNA sequencing and peptide analysis

A cDNA clone, pFL18, carrying a putative full-length fibroin light chain (L-chain) sequence was isolated and its nucleotide sequence was determined. This revealed the presence of an open reading frame corresponding to a polypeptide with 262 amino acid residues. The sequence was concluded to be that of the L-chain with its signal peptide because corresponding amino acid sequences for the seven tryptic and the four chymotryptic peptides from the purified L-chain were all included and an N-terminal region having typical properties of a signal peptide was present. The N terminus of the mature form of L-chain was identified as N-acetyl serine by analyzing the acyl-dansylhydrazide derived from the N-acyl-amino acid which had been released from the N-terminal blocked chymotryptic peptide by the acylamino acid-releasing enzyme. It was suggested that a signal peptide had cleaved between Pro18 and Ser19, yielding a mature L-chain polypeptide consisting of 244 amino acid residues. The molecular weight of the L-chain was calculated to be 25,800 including the N-acetyl group. The L-chain contained three Cys residues, two of which were suggested to form an intramolecular disulfide linkage, leaving the third one at the most C-terminal position and in a relatively hydrophilic region as the most probable site of disulfide linkage with the fibroin heavy chain.     

Isolation and characterization of a cDNA coding for human myeloperoxidase

A cDNA encoding the carboxyl-terminal fragment of the human myeloperoxidase heavy chain was isolated and characterized. It was then used to determine the locations of the myeloperoxidase light and heavy chains in the polypeptide precursor. A cDNA library from poly(A)+ RNA from human leukemia HL-60 cells was constructed in pBR322 and screened by differential hybridization with enriched and depleted cDNA probes and then by hybridization with an oligonucleotide probe. A cDNA clone containing 1278 bp with an open reading frame of 474 bp and a 3' noncoding region of 804 bp was isolated. The amino acid sequence deduced from the nucleotide sequence consisted of 158 residues including a sequence of 14 amino acids known to be present in the heavy chain of the molecule. The cDNA also included a stop codon of TAG followed by a noncoding sequence that included a potential recognition site for polyadenylylation and a poly(A) tail. RNA transfer blot analysis with the cDNA probe indicated that myeloperoxidase mRNA was approximately 3.3 kb in length. In vitro translation of the mRNA selected by cDNA hybridization revealed preferential synthesis of a 74,000-Da polypeptide precursor that could be precipitated with anti-myeloperoxidase IgG. Antibodies specific for the heavy and light chains of myeloperoxidase were isolated from antiserum by affinity chromatography employing Sepharose columns covalently bound to the heavy or light chains. Antibodies specific for the light chain or the heavy chain readily precipitated the 74,000-Da precursor polypeptide. These results indicated that myeloperoxidase is synthesized as a single chain which undergoes processing into a light and heavy chain. Furthermore, the heavy chain of myeloperoxidase originates from the carboxyl terminus of the precursor polypeptide.     

Human low-molecular-weight urinary urokinase. Partial characterization and preliminary sequence data of the two polypeptide chains

Low-molecular-weight urokinase (molecular weight 33100) was separated by analytical and preparative isoelectric focusing into five major subforms with isoelectric points between 8.7 and 9.6. These subforms are very similar in molecular weight, specific activity, amino acid composition and content of amino sugar and their N-terminal sequence constellation is identical. Low-molecular-weight urokinase consists of two polypeptide chains connected by a single disulfide bridge. The N-terminal region of the heavy chain (calculated Mr 30700) exhibits homology within the first 46 residues analyzed, with the known primary structure of other serine proteases. The mini chain (Mr 2426), whose complete sequence was determined, consists of 21 residues which show homology with the primary structure of the C-terminal region of the plasmin heavy chain. Based on sequence data and homology criteria with serine proteases a single-chain urokinase precursor is postulated having a peptide bond constellation between heavy and light chain region compatible with the requirements for serine protease activation.     

A novel form of the polypeptide PHI isolated in high yield from bovine upper intestine. Relationships to other peptides of the glucagon-secretin family

A novel form of the polypeptide termed PHI (peptide HI with N-terminal histidine and C-terminal isoleucine amide) has been isolated from bovine upper intestine. This bovine peptide was obtained in a 40 times higher yield than the corresponding polypeptide isolated from porcine intestine. Bovine PHI is, like porcine PHI, composed of 27 amino acid residues. The complete amino acid sequence of the bovine peptide is His-Ala-Asp-Gly-Val-Phe-Thr-Ser-Asp-Tyr-Ser-Arg-Leu-Leu-Gly-Gln-Leu-Ser- Ala- Lys-Lys-Tyr-Leu-Glu-Ser-Leu-Ile-NH2. This sequence differs from porcine PHI at position 10 and from human PHI at positions 10, 12 and 27. The amino acid residue exchange between porcine and bovine PHI makes the latter more similar to the vasoactive intestinal polypeptide (VIP), gastric inhibitory polypeptide (GIP), glucagon and the growth-hormone-releasing factor (GRF).     

Molecular cloning of the 18-kDa growth-associated protein of developing brain

An 18-kDa protein (designated herein as the heparin-binding growth-associated molecule, HB-GAM), the expression of which in rat brain correlates to the rapid postnatal developmental phase, was previously isolated and suggested to have a role in the maturation and growth of brain (Rauvala, H. (1989) EMBO J. 8, 2933-2941). A protein with a similar molecular mass, similar heparin-binding properties, and the same N-terminal sequence was more recently also isolated as a mitogen for NIH 3T3 cells (Milner, P. G., Li, Y.-T., Hoffman, R. M., Kodner, C. M., Siegel, N. R., and Deuel, T. F. (1989) Biochem. Biophys. Res. Commun. 165, 1096-1103). This study reports the cloning and sequencing of the cDNA that encodes HB-GAM. The sequence that precedes the structure of the mature molecule has the characteristics of a signal sequence found in secretory proteins. The sequence of HB-GAM is a novel structure that contains 136 amino acid residues. The sequence is very rich in cationic amino acids (24% of the residues); lysine cluster sequences are found in the N-terminal and C-terminal ends of the structure. Cysteine is also abundant in the sequence (7% of the residues). The only homologous sequence found in computer searches is the retinoic acid-induced differentiation factor. The mRNA of HB-GAM detected by the cloned cDNA shows the same kind of developmental regulation as the protein; the mRNA is strongly expressed during the early postnatal growth phase of rat brain as compared with embryonic or adult tissue.     

Amino acid sequence of bovine protein Z: a vitamin K-dependent serine protease homolog

The amino acid sequence of protein Z has been determined from sequence analysis performed on fragments obtained by chemical and enzymatic degradations. The polypeptide consists of a single chain containing 396 amino acid residues (Mr 43 677). Comparison with the vitamin K-dependent plasma proteins reveals an extensive homology. The N-terminal part, containing 13 gamma-carboxyglutamic acid and one beta-hydroxyaspartic acid residue, is extensively homologous to and of similar length to the light chain of factor X. The remainder of protein Z is homologous to the serine proteases and of similar size to the heavy chain of factor Xa, but of the active site residues only aspartic acid-102 is present. Histidine-57 and serine-195 are replaced in protein Z by threonine and alanine, respectively. The physiological function of protein Z is still uncertain.     

Cloning of cDNA encoding the membrane-bound form of bovine beta 1,4-galactosyltransferase

UDPgalactose: N-acetyl-D-glucosamine 4-beta-D-galactosyltransferase (EC 2.4.1.38) (GalT) is a Golgi-membrane-bound enzyme that participates in the biosynthesis of the oligosaccharide structures of glycoproteins and glycolipids. Synthetic DNA oligomers representing segments of the published partial cDNA sequence for bovine GalT were used as molecular probes to isolate from bovine-liver cDNA libraries overlapping cDNA clones that span 1728 nucleotides and potentially code for the entire polypeptide chain of bovine galactosyltransferase. The cDNA sequence for bovine GalT reveals a 1206-base-pair open reading frame that codes for 402 amino acids, including a presumptive N-terminal membrane anchoring domain of 20 hydrophobic amino acids. The colinearity between the cDNA sequence and 29 non-overlapping amino acid residues which were positively identified by N-terminal sequencing of two polypeptides isolated from the soluble form of the enzyme was consistent with the translation frame and confirmed the authenticity of the cDNA clones. The finding of an N-terminal hydrophobic segment which serves as the membrane anchor and signal sequence suggests that the C-terminal region of the GalT polypeptide is oriented within the lumen of the Golgi membranes. This conclusion is in agreement with previous biochemical studies which indicated that the 51-kDa and 42-kDa soluble forms of the enzyme which encompass the C-terminal 324 and 297 amino acid residues of the entire GalT polypeptide, respectively, include the catalytic site.     

Primary structure of the ovine pituitary follitropin beta-subunit.

The complete amino acids sequence of the ovine pituitary follitropin beta-subunit was established by studying the tryptic, chymotryptic and thermolytic peptides. The N-terminal sequence of the subunit was confirmed by subjecting the oxidated protein to Edman degradation in an automated sequenator. Automated Edman degradation of the reduced and alkylated (with iodo [14C]acetamide) beta-subunit indicated that most of the molecules used in the sequence studies had lost the N-terminal serine residue. This also confirmed the location of the first five half-cystine residues in the sequence. The proposed structure shows the presence of 111 amino acid residues with the two oligosaccharide moieties linked to asparagine residues located at positions 6 and 23. Heterogeneity occurs at both the termini of the polypeptide chain. Comparison of the sequence of beta-subunit of the ovine hormone with that proposed for human follitropin beta-subunit shows the absence of any deletions in the middle of the peptide chain. Of the 13 replacements, 11 residues can be explained on the basis of a single base change in the codon. The single tryptophan residue of the follitropin occupies an identical position in all the four species that have been studied. The region corresponding to residues 63-105 of the ovine beta-subunit is highly conserved in all the species.     

Molecular cloning and nucleotide sequences of the complementary DNAs to chicken skeletal muscle myosin two alkali light chain mRNAs.

We report here the molecular cloning and sequence analysis of DNAs complementary to mRNAs for myosin alkali light chain of chicken embryo and adult leg skeletal muscle. pSMA2-1 contained an 818 base-pair insert that includes the entire coding region and 5' and 3' untranslated regions of A2 mRNA. pSMA1-1 contained a 848 base-pair insert that included the 3' untranslated region and almost all of the coding region except for the N-terminal 13 amino acid residues of the A1 light chain. The 741 nucleotide sequences of A1 and A2 mRNAs corresponding to C-terminal 141 amino acid residues and 3' untranslated regions were identical. The 5' terminal nucleotide sequences corresponding to N-terminal 35 amino acid residues of A1 chain were quite different from the sequences corresponding to N-terminal 8 amino acid residues and of the 5' untranslated region of A2 mRNA. These findings are discussed in relation to the structures of the genes for A1 and A2 mRNA.     

Molecular cloning of mouse and bovine chondromodulin-II cDNAs and the growth-promoting actions of bovine recombinant protein

We previously determined the complete primary sequence of a heparin-binding growth-promoting factor, chondromodulin-II (ChM-II), which stimulated the growth of chondrocytes and osteoblasts in culture. Bovine ChM-II was a 16-kDa basic protein with 133 amino acid residues and exhibited a significant sequence similarity to the repeats of the chicken mim-1 gene product. Here we report the nucleotide sequences of bovine and mouse ChM-II cDNAs. The cDNAs each contained an open-reading frame corresponding to the ChM-II precursor with 151 amino acid residues. The N-terminus of the precursor included a secretory signal sequence of 18 amino acids prior to the mature ChM-II sequence. Unlike MIM-1, there was no repeat structure in the precursor protein, indicating that ChM-II was encoded as a gene product distinct from MIM-1. We then expressed recombinant bovine ChM-II protein which was purified to homogeneity. The recombinant protein stimulated the growth of rabbit growth plate chondrocytes, mouse MC3T3-E1 cells and rat UMR-106 osteoblastic cells in vitro.     

Sequencing and overexpression of the Escherichia coli aroE gene encoding shikimate dehydrogenase.

The Escherichia coli aroE gene encoding shikimate dehydrogenase was sequenced. The deduced amino acid sequence was confirmed by N-terminal amino acid sequencing and amino acid analysis of the overproduced protein. The complete polypeptide chain has 272 amino acid residues and has a calculated Mr of 29,380. E. coli shikimate dehydrogenase is homologous to the shikimate dehydrogenase domain of the fungal arom multifunctional enzymes and to the catabolic quinate dehydrogenase of Neurospora crassa.     

Isolation and expression of cDNA clones encoding mammalian poly(A) polymerase.

cDNA clones encoding mammalian poly(A) polymerase were isolated with probes generated by the polymerase chain reaction based on amino acid sequences derived from the purified enzyme. A bovine cDNA clone was obtained encoding a protein of 82 kDa. Expression in Escherichia coli resulted in the appearance of a poly(A) polymerase activity that was dependent on the addition of the purified specificity factor CPF and the presence of the polyadenylation signal AAUAAA in the RNA substrate. The activity copurified with a polypeptide of the expected size. A second class of cDNAs encoded a polypeptide of 43 kDa which was closely related to the N-terminal half of the 82 kDa protein. Northern blots showed two mRNAs of 4.2 and 2.4 kb that probably correspond to the two classes of cDNAs, as well as a third band of 1.3 kb. The sequence of the N-terminal half of bovine poly(A) polymerase is 47% identical with the amino acid sequence of the corresponding part of yeast poly(A) polymerase. Homologies to other proteins are of uncertain significance.     

Complete amino acid sequence of the A chain of human complement-classical-pathway enzyme C1r.

The amino acid sequence of human C1r A chain was determined, from sequence analysis performed on fragments obtained from C1r autolytic cleavage, cleavage of methionyl bonds, tryptic cleavages at arginine and lysine residues, and cleavages by staphylococcal proteinase. The polypeptide chain has an N-terminal serine residue and contains 446 amino acid residues (Mr 51,200). The sequence data allow chemical characterization of fragments alpha (positions 1-211), beta (positions 212-279) and gamma (positions 280-446) yielded from C1r autolytic cleavage, and identification of the two major cleavage sites generating these fragments. Position 150 of C1r A chain is occupied by a modified amino acid residue that, upon acid hydrolysis, yields erythro-beta-hydroxyaspartic acid, and that is located in a sequence homologous to the beta-hydroxyaspartic acid-containing regions of Factor IX, Factor X, protein C and protein Z. Sequence comparison reveals internal homology between two segments (positions 10-78 and 186-257). Two carbohydrate moieties are attached to the polypeptide chain, both via asparagine residues at positions 108 and 204. Combined with the previously determined sequence of C1r B chain [Arlaud & Gagnon (1983) Biochemistry 22, 1758-1764], these data give the complete sequence of human C1r.