Structure of a cDNA for the pro alpha 2 chain of human type I procollagen. Comparison with chick cDNA for pro alpha 2(I) identifies structurally conserved features of the protein and the gene

Nucleotide sequences were determined for cloned cDNAs encoding for more than half of the pro alpha 2 chain of type I procollagen from man. Comparisons with previously published data on homologous cDNAs from chick embryos made it possible to examine evolution of the gene in two species which have diverged for 250-300 million years. The amino acid sequence of the alpha-chain domain supported previous indications that there is a strong selective pressure to maintain glycine as every third amino acid and to maintain a prescribed distribution of charged amino acids. However, there is little apparent selective pressure on other amino acids. The amino acid sequence of the C-propeptide domain showed less divergence than the alpha-chain domain. The 5' end or N terminus of the human C-propeptide, however, contained an insert of 12 bases coding for 4 amino acids not found in the chick C-propeptide. About 100 amino acid residues from the N terminus, two residues found in the chick sequence were missing from the human. In the second half of the C-propeptide, there was complete conservation of a 37 amino acid sequence and conservation of 50 out of 51 amino acids in the same region, an observation which suggested that the region serves some special purpose such as directing the association of one pro alpha 2(I) C-propeptide with two pro alpha 1(I) C-propeptides so as to produce the heteropolymeric structure of type I procollagen. In addition, comparison of human and chick DNAs for pro alpha 2(I) revealed three different classes of conservation of nucleotide sequence which have no apparent effect on the structure of the protein: a preference for U on the third base position of codons for glycine, proline, and alanine; a high degree of nucleotide conservation in the 51 amino acid highly conserved region of the C-propeptide; a high degree of nucleotide conservation in the 3'-noncoding region. These three classes of nucleotide conservation may reflect unusual features of collagen genes, such as their high GC content or their highly repetitive coding sequences.     

Isolation of tropoelastin a from lathyritic chick aortae.

Tropoelastin a was isolated from lathyritic chick aortae by using severe denaturing conditions for the initial extraction. The amino acid composition of this new species of tropoelastin is elastin-like in its high proportion of proline, glycine, alanine and valine. However, it differs from authentic tropoelastin b in containing a higher percentage of polar amino acids and cysteine residues. In addition, the amount of proline hydroxylation is 3 times higher than that found in chick tropoelastin b.     

Complete amino acid sequence of 14 kDa beta-galactoside-binding lectin of chick embryo

The complete amino acid sequence of a soluble beta-galactoside-binding lectin (subunit MW 14,500) of chick embryo was determined. The protein consists of 134 amino acids beginning with serine and ending with glutamic acid, and its N-terminal was blocked with acetate. The agreement of the present result with that obtained from nucleotide sequence analysis (Y. Ohyama et al. (1986) Biochem. Biophys. Res. Commun. 134, 51-56) indicates the lack of a cleavable leader sequence. Internal homologies were observed in several regions along the polypeptide chain. The highest homology (55% identity) was found between residues 42-58 and residues 112-128. This suggests that chick 14 kDa lectin may have evolved via several gene duplications.     

The structure of anchorin CII, a collagen binding protein isolated from chondrocyte membrane

cDNA clones for anchorin CII (Mr = 34,000), a collagen-binding protein, were isolated from a lambda gt 11 cDNA library prepared from chick cartilage mRNA. Several overlapping clones were characterized which gave rise to an open reading frame coding for 329 residues and a 3'-untranslated segment of 500 base pairs. The clones were identified as coding for anchorin by hybrid select translation analysis and by comparing the deduced amino acid sequence with the sequence of 10 tryptic peptides of the protein. A hydrophobic domain of 25 residues interrupted with 3 polar residues was identified with the carboxyl-terminal portion. There was no evidence for an aminoterminal signal peptide. Northern analysis revealed that the 5' probe hybridizes to a single 1.7-kilobases (kb) mRNA species, whereas the 3' probe hybridizes to two mRNA species of 1.7 kb and 5 kb, which are present in many cells including chondrocytes, crop cells, and fibroblasts. The level of anchorin mRNA in chick embryo fibroblasts was increased by infection with Rous sarcoma virus.     

Structural analysis of homologous repeated domains in alpha-actinin and spectrin

The amino acid sequences of chick and slime mould alpha-actinin each contain four repeats of approximately 122 residues. These repeats are homologous to the 18-22 repeats, each of approximately 106 residues, found in the alpha and beta subunits of spectrin and fodrin, and to the multiple repeats of approximately 110 residues found in the Duchenne muscular dystrophy protein (dystrophin). The repeats correspond to the elongated rod-like portion of these molecules. We present a multiple sequence alignment of 21 repeats from this superfamily (8 alpha-actinin and 13 spectrin/fodrin), based on optimal pairwise alignments, from which a characteristic consensus pattern of amino acid types is deduced. Trp 46 is invariant in all but one repeat, and physicochemical classes of amino acids are conserved at 25 other positions. Secondary structure prediction on both the alpha-actinin and spectrin repeats taken together with the distribution of proline residues in the sequences, strongly suggest that each repeated domain consists of a four-helix structure. Our predictions differ significantly from previous three-helix models based on analyses of fewer sequences. To determine possible interdomain regions, sites of limited proteolysis of the native chick alpha-actinin dimer were determined and located in the amino acid sequence. The majority of these sites were in corresponding positions in different repeats within a segment predicted as a long helix. We propose a model, consistent with the overall dimensions of the rod-like portions of the molecules, in which these long, probably interrupted helices, link adjacent domains.     

Characterization of an associated microfibril protein through recombinant DNA techniques.

The complete primary structure of a new extracellular protein associated with elastic fiber microfibrils was determined by recombinant DNA techniques. Antiserum to insoluble bovine ocular zonule protein was used to screen a lambda gt11 cDNA expression library constructed from whole chick embryo poly(A)+ RNA. The cDNAs encoding immunoreactive fusion polypeptides were then used to rescreen the library by plaque hybridization. Nucleotide sequencing of overlapping cDNA clones revealed an open translation reading frame of 1326 bases beginning at an initiation start sequence and ending at a stop codon. The contiguous cDNA sequence contains a 3'-untranslated region of 563 bases with a possible polyadenylation site 16 bases upstream from the poly(A) tail. Primer extension of chick aortic mRNA taken together with the sequence data, reveals a 5'-untranslated region of 95 bases extending upstream from the translation start site. Northern blot analyses indicated that the isolated cDNA hybridized with a 2.1-kilobase mRNA in preparations of whole chick embryo and chick embryonic aortic, heart, and muscle RNAs. The initial translation protein encoded by the cDNA is 53,932 kDa and possesses a hydrophilic amino acid composition with glutamic acid comprising 22% of the total amino acid residues. Antiserum was elicited to a synthetic peptide sequence (14 amino acids) encoded within the deduced protein primary structure. Western blots of extracted proteins from chick embryonic aortae cultured in the presence of beta-aminopropionitrile showed that the medium and a mild salt extract contained an immunoreactive protein possessing an apparent molecular mass of 58,000 whereas harsh denaturants extracted a 32,000-kDa protein. Pulse-chase experiments using radiolabeled lysine showed that the newly synthesized 58,000-kDa protein was chased into a 32,000-kDa protein within a 2-24-h period. Immunoelectron microscopy of tissue sections from chick aortae, bovine nuchal ligament, and human ocular zonules showed that the peptide-elicited antibody localized specifically to ultrastructurally definable microfibril structures.     

Brain natriuretic peptide-32: N-terminal six amino acid extended form of brain natriuretic peptide identified in porcine brain

Brain natriuretic peptide (BNP) is a newly identified peptide of 26 residues, which has a remarkable homology to but is distinct from atrial natriuretic peptide. The peptide exerts natriuretic-diuretic activity as well as potent chick rectum relaxant activity. By using radioimmunoassay specific to BNP and immunoaffinity chromatography, we have isolated from porcine brain a novel peptide of 32 residues carrying a BNP structure at the C-terminus. The amino acid sequence of this peptide was determined to be: Ser-Pro-Lys-Thr-Met- Arg-Asp-Ser-Gly-Cys-Phe-Gly-Arg-Arg-Leu-Asp-Arg-Ile-Gly-Ser-Leu-Ser-Gly- Leu- Gly-Cys-Asn-Val-Leu-Arg-Arg-Tyr. This peptide is an N-terminal six amino acid extended form of BNP and henceforth is designated BNP-32. BNP and BNP-32 are found to be major forms of BNP family in porcine brain.     

Isolation and characterization of a vinculin cDNA from chick-embryo fibroblasts.

A chick-embryo fibroblast lambda gt11 cDNA library was screened with affinity-purified antibodies to chick gizzard vinculin. One recombinant was purified to homogeneity and the fusion protein expressed in Escherichia coli strain C600. The fusion protein was unstable, but polypeptides that reacted with vinculin antibodies, but not non-immune immunoglobulin, were detected by Western blotting. The recombinant contained a single EcoRI fragment of 2891 bp with a single open reading frame. The deduced protein sequence could be aligned with that of six CNBr-cleavage peptides and two tryptic peptides derived from chicken gizzard vinculin. AUG-247 has tentatively been identified as the initiation codon, as it is contained within the consensus sequence for initiation sites of higher eukaryotes. The cDNA lacks 3' sequence and encodes 74% of the vinculin sequence, presuming the molecular mass of vinculin to be 130,000 Da. Analysis of the deduced sequence showed no homologies with other protein sequences, but it does display a triple internal repeat of 112 amino acid residues covering residues 259-589. The sequences surrounding the seven tyrosine residues in the available sequence were aligned with the tyrosine autophosphorylation consensus sequence found in protein tyrosine kinases. Tyr-822 showed a good match to this consensus, and may represent one of the two major sites of tyrosine phosphorylation by pp60v-sre. Northern blots showed that the 2.89 kb vinculin cDNA hybridized to one size of mRNA (approx. 7 kb) in chick-embryo fibroblasts, chick smooth muscle and chick skeletal muscle. Southern blots revealed multiple hybridizing bands in genomic DNA.     

Isolation and partial characterization of the amino-terminal propeptide of type II procollagen from chick embryos

The amino-terminal propeptide from type II procollagen was isolated from organ cultures of sternal cartilages from 17-day-old chick embryos. The procedure provided the first isolation of the propeptide in amounts adequate for chemical characterization. The propeptide had an apparent molecular weight of 18000 as estimated by gel electrophoresis in sodium dodecyl sulfate. It contained a collagen-like domain as demonstrated by its amino acid composition, circular dichroism spectrum, and susceptibility to bacterial collagenase. One residue of hydroxylysine was present, the first time this amino acid has been detected in a propeptide. The peptide contained no methionine and only two residues of half-cystine. Antibodies were prepared to the propeptide and were used to establish its identity. The antibodies precipitated type II procollagen but did not precipitate type II procollagen from which the amino and carboxy propeptides were removed with pepsin. Also, they did not precipitate the carboxy propeptide of type II procollagen. The data demonstrated th at the type II amino propeptide was similar to the amino propeptides of type I and type III procollagens in that it contained a collagen-like domain. It differed, however, in that it lacked a globular domain as large as the globular domain of 77-86 residues found at the amino-terminal ends of the pro alpha 1 chains of type I and type III procollagens.     

Monoclonal antibody studies of creatine kinase. The ART epitope: evidence for an intermediate in protein folding.

Chemical cleavage at cysteine residues with nitrothiocyanobenzoic acid shows that the last 98 amino acids of the 380-amino-acid sequence of chick muscle creatine kinase are sufficient for binding of the monoclonal antibody CK-ART. Removal of the last 30 amino acids by cleavage at methionine residues with CNBr results in loss of CK-ART binding. CK-ART binding is also lost when these C-terminal methionine residues are oxidized to sulphoxide, but binding is regained on reduction. Proteinase K 'nicks' native CK at a single site near the C-terminus and two fragments of 327 amino acides and 53 amino acids can be separated by subsequent SDS or urea treatment. CK-ART still binds normally to 'nicked' CK, which is enzymically inactive. After treatment with either urea (in a competition enzyme-linked immunosorbent assay) or SDS (on Western blots), however, CK-ART binds to neither of the two fragments, although these treatments do not affect binding to intact CK. This suggests that parts of both CK fragments contribute to the CK-ART epitope. CK-ART is both species- and isoenzyme-specific, binding only to chick M-CK. The only C-terminal regions containing chick-specific sequences are residues 300-312 and residues 368-371, the latter group being close to the essential methionine residues. We suggest that one, or possibly both, of these regions is involved in forming the conformational epitope on the surface of the CK molecule which CK-ART recognizes. Native CK is resistant to trypsin digestion. The C-terminal half of urea-treated and partly-refolded CK is also resistant to trypsin digestion, whereas the N-terminal half is readily digested. The results suggest a C-terminal region which can refold more rapidly than the rest of the CK molecule and provide evidence for an intermediate in CK refolding.     

Synthesis of atrial natriuretic peptides and studies on structural factors in tissue specificity

Two atrial natriuretic peptides, containing 25 amino acid residues, ANF IV, and 21 amino acid residues, ANF V, were synthesized by a solid phase method and oxidized with K3Fe(CN)6 to form a disulfide bridge. Synthetic ANF IV exhibited a natriuretic activity with an ED50 70 times higher than that of synthetic ANF V, whereas the longer peptide was only 2.5 times more potent in chick rectal smooth muscle relaxant activity. Both peptides inhibited norepinephrine-induced contraction of rabbit aorta. The shorter peptide, ANF V, was 300 times less efficient than the longer peptide, ANF IV. It is proposed that the carboxy-terminal of ANF IV seems to have a modulating effect on receptor affinity in kidney and vascular tissue.     

Complete amino acid sequence of a beta-galactoside-binding lectin from human placenta

The complete amino acid sequence of a beta-galactoside-binding lectin from human placenta was determined at protein level. The lectin consists of 134 amino acids and its N-terminal alanine is blocked with acetate. The lectin shows about 50% similarity with chick 14K lectin, which was the first vertebrate beta-galactoside-binding lectin completely sequenced. Only 14 residues proved to be different from those of rat lung lectin, the sole mammalian lectin of which the complete sequence has been reported.     

Sequence of carbonic anhydrase II cDNA from chick retina

Sequences of three cDNA clones for carbonic anhydrase II (CA-II) from chick retina are presented. The longest cDNA clone encodes all but the first three amino acids of CA-II, and the encoded sequence generally agrees with published fragments of CA-II sequence from chick red blood cells. It is 70% identical to human CA-II; the active-site residues are conserved, but the chick protein has six extra cysteines. There is a long 3'-untranslated region which contains a second open reading frame, but this is not conserved. There appears to be a single CA-II gene in the chick. Some anomalies in cDNA synthesis and in Bal31 deletion are noted.     

Molecular cloning and expression of nitric oxide synthase gene in chick embryonic muscle cells

The chick skeletal muscle nitric oxide synthase (NOS) gene was cloned in order to further define the involvement of NOS in the differentiation of skeletal muscle cells. The respective cDNA had an open reading frame of 1136 amino acid residues, predicting a protein of 129,709.85 Da, and recognition sites for FAD, FMN, NADPH, and a calmodulin-binding site like those of other mammalian NOS's. Alignment of the deduced amino acid sequence revealed high homology with mammalian inducible NOS (iNOS), but not other NOS isoforms, suggesting chick skeletal muscle NOS may be an iNOS isoform. Immunoblots showed that NOS expression was highly restricted in embryonic muscle, but not in adult skeletal muscle: NOS expression markedly increased from embryonic day 9, reached a maximum by embryonic day 13, and then gradually declined until it was no longer detectable on embryonic day 19. When muscle cells obtained on embryonic day 12 were cultured, NOS expression increased transiently prior to the onset of differentiation and decreased thereafter. Inhibition of NOS expression by PDTC completely prevented muscle cell differentiation, as indicated by the inhibition of expression of myosin heavy chain and creatine kinase. The inhibitory effect of PDTC was completely reversed by addition of sodium nitroprusside, a compound that produces NO. These results clearly indicate that NOS is significantly involved in the differentiation of chick skeletal muscle cells.     

Covalent structure of collagen: amino acid sequence of alpha 2-CB5 of chick skin collagen containing the animal collagenase cleavage site.

The amino acid sequence of the 112 residues from the amino terminus of alpha 2-CB5 from chick skin collagen was determined by automated sequential degradation of intact alpha 2-CB5 and several chymotryptic and tryptic peptides. This segment of the peptide includes the site of the action of animal collagenases. As compared to the sequence around the alpha 1 cleavage site, the alpha 2 sequence is notable for the remarkable constancy of the residues to the amino side and the relative abundance of hydrophobic residues to the carboxyl side of the cleavage site, suggesting that these features are important in the recognition by the enzyme. The sequence of this region of the alpha 2 chain is consistent with the Gly-X-Y triplet structure and the preference of certain residues for either the X or Y position in distribution. However, three of the six residues of leucine were found in the Y position rather than the X position. Leucine residues were found only once in the Y position in the alpha 1 (I) chain. This preference does not appear to hold in the alpha 2 chain.     

The amino acid sequence of equine milk lysozyme

The amino acid sequence of equine milk lysozyme has been elucidated. The study involves the determination of the sequence of the N-terminal region of the whole protein, cyanogen bromide fragments, tryptic and chymotryptic peptides and fragments produced by chemical cleavage after tryptophan residues. The protein consists of a single chain of 129 amino acid residues and has a Mr of 14647. While equine milk lysozyme has the essential features of a c(chick)-type lysozyme, there is only 51% sequence homology with human milk lysozyme and 50% with domestic hen egg white lysozyme. Some of the implications of the large number of differences are discussed.     

Comparison between avian and human prolyl 4-hydroxylases: studies on the holomeric enzymes and their constituent subunits.

Prolyl 4-hydroxylase, a key enzyme in collagen biosynthesis, catalyzes the conversion of selected prolyl residues to trans-hydroxyproline in nascent or completed pro-alpha chains of procollagen. The enzyme is a tetramer composed of two nonidentical subunits, designated alpha and beta. To compare the enzyme and its subunits from different sources, the chick embryo and human placental prolyl 4-hydroxylases were purified to homogeneity and their physicochemical and immunological properties were determined. Both enzymes were glycoproteins with estimated apparent molecular weights ranging between 400 and 600 kDa. Amino acid and carbohydrate analyses showed slight differences between the two holomeric enzymes, consistent with their deduced amino acid sequences from their respective cDNAs. Human placental prolyl 4-hydroxylase contained more tightly bound iron than the chick embryo enzyme. Immunodiffusion of the human placental enzyme with antibodies raised against the purified chick embryo prolyl 4-hydroxylase demonstrated partial identity, indicating different antigenic determinants in their tertiary structures. The enzymes could be separated by high-resolution capillary electrophoresis, indicating differential charge densities for the native chick embryo and human placental proteins. Electrophoretic studies revealed that the human prolyl 4-hydroxylase is a tetrameric enzyme containing two nonidentical subunits of about 64 and 62 kDa, in a ratio of approximately 1 to 2, designated alpha and beta, respectively. In contrast, the chick embryo alpha and beta subunit ratio was 1 to 1. Notably, the human alpha subunit was partially degraded when subjected to electrophoresis under denaturing conditions. Analogously, when the chick embryo enzyme was subjected to limited proteolysis, selective degradation of the alpha subunit was observed. Finally, only the alpha subunit was bound to Concanavalin A demonstrating that the alpha subunits of prolyl 4-hydroxylase in both species were glycosylated. Using biochemical techniques, these results demonstrated that the 4-trans-hydroxy-L-proline residues in human placental collagens are synthesized by an enzyme whose primary structure and immunological properties differ from those of the previously well-characterized chick embryo enzyme, consistent with their recently deduced primary structures from cDNA sequences.