Identification of hormonogenic domains in the carboxyl terminal region of bovine thyroglobulin

A segment of 986 nucleotides corresponding to the 3' end of the 8.5 kb bovine thyroglobulin (Tg) mRNA has been sequenced. An open reading frame of 302 codons was found, ending with TGA and preceding an 80 nucleotide long 3' untranslated sequence. The encoded protein sequence provided the first data on the carboxyl terminal portion of Tg. Lysine was identified as the last residue. Comparison of the amino acid sequence with that of peptides known to contain thyroid hormones in the mature protein, lead to the identification of three regions involved in thyroid hormone formation. Two closely linked thyroxine- forming sites were found 182 and 196 amino acids from the carboxyl terminus respectively. The antepenultimate amino acid of the protein corresponded to the recently described triiodothyronine-forming site. Together with the previous localization of the main thyroxine-containing peptide at the amino terminus, the present results provide a map of all hormonogenic sites identified to date in Tg.     

Presence of hormonogenic and repetitive domains in the first 930 amino acids of bovine thyroglobulin as deduced from the cDNA sequence

The sequence of the first 2831 nucleotides of bovine thyroglobulin mRNA has been determined from the analysis of a cDNA clone. Following a 41-nucleotide 5' untranslated sequence, a single open-reading frame encoding 930 amino acids was observed. This corresponds to the aminoterminal third of thyroglobulin, preceded by a putative signal peptide of 19 amino acids. The protein sequence was found to be essentially made of the sevenfold repetition of a 60-amino-acid-long building unit, interrupted at fixed positions by unrelated segments of variable length. The presence of an internal homology within the repetitive unit itself suggests that the 5' region of the thyroglobulin gene has evolved from the initial duplication of a relatively short sequence, followed by the serial duplication of the resulting unit. The tyrosine residue at position five has been assigned an important hormonogenic function [Mercken, L., Simons, M.-J. and Vassart, G. (1982) FEBS Lett. 149, 285-287]. This residue is flanked by sequence elements related to the repeated unit, suggesting that the hormonogenic domain evolved also from the basic ancestor sequence.     

Primary structure of the gene for the murine Ia antigen-associated invariant chains (Ii). An alternatively spliced exon encodes a cysteine-rich domain highly homologous to a repetitive sequence of thyroglobulin.

The gene for murine Ia-associated invariant (Ii) chains (Ii31 and Ii41) was characterized by sequence analysis. The gene extends over approximately 9 kb and is organized in nine exons. Exon 1 encodes the 5' untranslated region and the cytoplasmic segment, exon 2 the membrane spanning segment and adjacent amino acids and exons 3-8 the extracytoplasmic portion of Ii31. Putative promoter sequences were found upstream of the start of the coding sequence. Between exons 6 and 7 an additional, alternatively spliced exon 6b has been identified. This exon is spliced into the mRNA coding for the Ii-related Ii41 protein. Exon 6b encodes a cysteine-rich domain of 64 amino acids. It shows a remarkably high homology to the repetitive elements in thyroglobulin, a precursor for thyroid hormone. Based on this homology, it is suggested that this domain (TgR) in Tg and in Ii41 may play a role either in hormone formation or as a carrier in the transport of molecules (thyroid hormone or processed antigen respectively) between intracellular compartments.     

Primary structure of human thyroglobulin deduced from the sequence of its 8448-base complementary DNA

The mRNA encoding human thyroglobulin has been cloned and sequenced. It is made up of a 8301-nucleotide segment encoding a preprotein monomer of 2767 amino acids, flanked by non-coding 5' and 3' regions of 41 and 106 nucleotides, respectively. This preprotein consists of a leader sequence of 19 amino acids, followed by the sequence of the mature monomer, corresponding to a polypeptide of 2748 amino acids (Mr = 302773). On its amino-terminal side, 70% of the monomer is characterized by the presence of three types of repetitive units. In contrast, the remaining 30% of the protein is devoid of repetitive units. This last region however shows an interesting homology (up to 64%) with the acetylcholinesterase of Torpedo californica. The sites of thyroid hormones synthesis are clustered at both ends of the thyroglobulin monomer. By contrast, the potential glycosylation sites are scattered along the polypeptide chain.     

The structure of a naturally occurring 10K polypeptide derived from the amino terminus of bovine thyroglobulin

A combination of data derived from peptide sequencing and nucleic acid sequencing of cloned cDNA fragments has been used to define the complete amino acid sequence of a 10,000 M.W., thyroxine containing polypeptide derived from bovine thyroglobulin. This fragment, TG-F, which was obtained following reduction and alkylation, has been placed at the amino terminus of the parent protein with hormone located at residue 5 in the primary sequence of the thyroglobulin molecule. The carboxyl terminal sequence of this fragment -Cys-Gln-Leu-Gln is found on the N-terminal side of a lys residue, suggesting that the peptide bond cleavage which occurs to produce this 80 residue fragment from the parent (330K) thyroglobulin chain is a gln-lys. In addition, the amino acid sequence of this 10K fragment contains: No sequence which would be a substrate for glycosylation and no carbohydrate. Several repeated homologous amino acid sequences. A striking number of beta-bends predicted from Chou-Fasman analyses, particularly near its carboxyl terminus.     

Surface location and high affinity for calcium of a 500-kd liver membrane protein closely related to the LDL-receptor suggest a physiological role as lipoprotein receptor.

We describe a cell surface protein that is abundant in liver and has close structural and biochemical similarities to the low density lipoprotein (LDL) receptor. The complete sequence of the protein containing 4544 amino acids is presented. From the sequence a remarkable resemblance to the LDL-receptor and epidermal growth factor (EGF) precursor is apparent. Three types of repeating sequence motifs entirely account for the extracellular domain of the molecule. These are arranged in a manner resembling four copies of the ligand binding and the EGF-precursor homologous region of the LDL-receptor. Following a proline-rich segment of 17 amino acids are found six consecutive repeats with close homology to EGF. A single membrane-spanning segment precedes a carboxy-terminal 'tail' of 100 amino acids. This contains two seven-amino acid sequences with striking homology to the cytoplasmic tail of the LDL-receptor in the region that contains the signal for clustering into coated pits. The mRNA for this protein is most abundant in liver, brain and lung. By using an antibody raised against a 13-amino acid peptide corresponding to the deduced amino acid sequence of the carboxy-terminus of the protein we have demonstrated its existence on the cell surface and its abundance in liver. Like the LDL-receptor this protein also strongly binds calcium, a cation absolutely required for binding of apolipoproteins B and E to their receptors. We propose that this LDL-receptor related protein (LRP) is a recycling lipoprotein receptor with possible growth-modulating effects.     

Primary structure of acetylcholinesterase: implications for regulation and function

A cDNA encoding acetylcholinesterase (AChE) (EC 3.1.1.7) from Torpedo californica was isolated and from its nucleotide sequence the entire amino acid sequence of the processed protein and a portion of the leader peptide has been deduced. Approximately 70% of the tryptic peptides from the catalytic subunit of the 11 S form have been sequenced, and a comparison of the peptide sequences with the sequence inferred from the cDNA suggests that the cDNA sequence derives from mRNA for the 11 S form of the enzyme. The amino acid sequence is preceded by a hydrophobic leader peptide and contains an open reading frame encoding for 575 amino acids characteristic of a secreted globular protein. Eight cysteines, most of which are disulfide linked, are found along with four potential sites of N-linked glycosylation. The active-site serine is located at residue 200. Local homology is found with other serine hydrolases in the vicinity of the active site, but the enzyme shows striking global homology with the COOH-terminal portion of thyroglobulin. Further comparison of the amino acid sequences of the individual enzyme forms with other cDNA clones that have been isolated should resolve the molecular basis for polymorphism of the AChE species.     

Rat apolipoprotein E mRNA. Cloning and sequencing of double-stranded cDNA

A 900-base pair clone corresponding to rat liver apolipoprotein E (apo-E) mRNA, and containing a 3'-terminal poly(A) segment, was identified from a library of rat liver cDNA clones in the plasmid pBR322 by specific hybrid selection and translation of mRNA. A restriction endonuclease DNA fragment from this recombinant plasmid was used to clone the 5'-terminal region of the apo-E mRNA by primed synthesis of cDNA. A portion of the double-stranded cDNA corresponding to the 3'-terminal region of apo-E mRNA was subcloned into the bacteriophage M13mp7 and employed as a template for the synthesis of a radioactively labeled, cDNA hybridization probe. This cDNA probe was used in a RNA-blot hybridization assay that showed the length of the apo-E mRNA to be about 1200 nucleotides. The hybridization assay also demonstrated that apo-E mRNA is present in rat intestine, but at about a 100-fold lower level than that of the rat liver. The nucleotide sequence of rat liver apo-E mRNA was determined from the cloned, double-stranded cDNAs. The amino acid sequence of rat liver apo-E was inferred from the nucleotide sequence, which showed that the mRNA codes for a precursor protein of 311 amino acids. A comparison to the NH2-terminal amino acid sequence of rat plasma apo-E indicated that the first 18 amino acids of the primary translation product are not present in the mature protein and are probably removed during co-translational processing. The coding region was flanked by a 3'-untranslated region of 109 nucleotides, which contained a characteristic AAUAAA sequence that ended 13 nucleotides from a 3'-terminal poly(A) segment. At the 5'-terminal region of the mRNA, 23 nucleotides of an untranslated region were also determined. The inferred amino acid sequence of mature rat apo-E, which contains 293 amino acids, was compared to the amino acid sequence of human apo-E, which contains 299 amino acids. Using an alignment that permitted a maximum homology of amino acids, it was found that overall, 69% of the amino acid positions are identical in both proteins. The amino acid identities are clustered in two broad domains separated by a short region of nonhomology, an NH2-terminal domain of 173 residues where 80% are identical, and a COOH-terminal domain of 84 residues where 70% are identical. These two domains may be associated with specific functional roles in the protein.     

水稻矮缩病毒第11号组分基因序列和编码蛋白的功能分析

水稻矮缩病毒(Rice Dwarf Virus-RDV)广泛分布于中国、日本及东南亚地区,侵染水稻和禾本科其它一些作物,是造成水稻减产的主要原因之一,对农作物危害极大。RDV属于呼肠孤病毒科(Re-oviridae)中的植物呼肠孤病毒属(Phytoreovirus)成员,其病毒粒子直径70nm,为20面体,有双层     

Wound-induced proteinase inhibitors from tomato leaves. I. The cDNA-deduced primary structure of pre-inhibitor I and its post-translational processing

A cDNA containing the coding region for the complete amino acid sequence of wound-induced proteinase Inhibitor I from tomato leaves was constructed in the plasmid pUC9 and characterized. The open reading frame codes for a protein of 111 amino acids. This deduced amino acid sequence revealed the presence of a 42-amino acid N-terminal sequence that is not found in the native protein. This sequence appears to contain a 23-amino acid segment typical of a signal sequence followed by a 19-amino acid sequence containing 9 charged amino acids. The 42-amino acid sequence is apparently lost during maturation to the native Inhibitor I and represents 38% of the translated protein. The Inhibitor I amino acid sequence contains 71% identity with potato tuber Inhibitor I sequence and 35% identity with an inhibitor from the leech.     

Thyroid hormonogenesis. Identification of a sequence containing iodophenyl donor site(s) in calf thyroglobulin

The formation of dehydroalanine in thyroglobulin is the result of the side chain elimination of an iodophenyl group during the thyroid hormone formation from two iodotyrosyl residues. This amino acid is easily converted to labeled alanine (upon reduction with [3H] borohydride) or changed to labeled aspartic acid (upon addition of Na14CN and subsequent acid hydrolysis). The cleavage of the protein by CNBr produced many stainable electrophoretic bands, but the autoradiography indicated the presence of a much smaller number of radioactive species. Although three major species raised attention, because they could be all jointly labeled and were present in all preparations, only a species of 15,900 Da was fully studied. It was isolated and its sequence partially determined by Edman degradation. It was established that this species corresponded to the thyroglobulin fragment between methionines 2,432 and 2,578. This peptide contains two hormonogenic sites (positions 2,555 and 2,569) which are either tyrosyl residues or hormone residues arising from them, and five additional tyrosines all potentially involved as donor sites in the hormonogenesis. Upon treatment with N-chlorosuccinimide, the fragment was split into three smaller peptides of about 2,900, 8,500, and 4,600 Da containing 1, 2, and 2 tyrosyl residues, respectively. Only the 8,500-Da subfragment contained [3H]Ala. This finding strongly suggests that at least some of the tyrosines involved as donor sites in thyroid hormonogenesis are within this peptide and possibly map at positions 2,469 and/or 2,522. Moreover, at minimum levels of iodination, when thyroglobulin contains the lowest number of hormone molecules, dehydroalanine is mostly found in the 15,900-Da peptide.     

Nucleotide sequence of human rotavirus genome segment 10, an RNA encoding a glycosylated virus protein.

The complete nucleotide sequence of human rotavirus (Wa strain) genome segment 10 was determined by using a cloned DNA copy. The sequence data indicated that segment 10 is A + T rich (65%) and consists of 750 base pairs. The positive strand of segment 10 contains a single open reading frame that extends 175 codons from the first AUG triplet (residues 42 through 44). The amino acid sequence of the segment 10 product was deduced from the nucleotide sequence. There are two distinct glycosylation sites at the N-terminal hydrophobic region, consistent with previous findings that this protein exists in a glycosylated form. The apparent molecular weight (20,000) of the unglycosylated, precursor polypeptide is in good agreement with the one calculated from the predicted amino acid sequence. Structural analysis of the positive strand (mRNA from segment 10) showed that it could form, like mRNA from segment 11, a stable panhandle structure involving the 5' and 3'-terminal regions. The nucleotide sequence of segment 10 from simian rotavirus, recently determined by Both et al. (J. Virol. 48:335-339, 1983) was found to be highly homologous to, and to share several important features with, segment 10 of human rotavirus.     

A novel human apolipoprotein (apoM).

A novel human apolipoprotein designated apolipoprotein M (apoM) is described. The unique N-terminal amino acid sequence of apoM was found in an approximately 26-kDa protein present in a protein extract of triglyceride-rich lipoproteins (TGRLP). The isolated apoM cDNA (734 base pairs) encoded a 188-amino acid residue-long protein, distantly related to the lipocalin family. The mRNA of apoM was detected in the liver and kidney. Western blotting demonstrated apoM to be present in high density lipoprotein (HDL) and to a lesser extent in TGRLP and low density lipoproteins (LDL). The first 20 amino acid residues of apoM constituted a hydrophobic segment with characteristic features of a signal peptide. This was retained in the mature protein because of the lack of a signal peptidase cleavage site. In vitro translation in the presence of microsomes demonstrated translocation of apoM over the membrane and glycosylation but no signal peptide cleavage. The in vitro translated product remained associated with the microsomes after treatment with carbonate at pH 11, demonstrating that apoM is an integral protein. This finding suggests that apoM is linked to the single phospholipid layer of lipoproteins with a hydrophobic signal anchor. In conclusion, a novel human apolipoprotein, the function of which remains to be determined, is described.     

Influenza B virus genome: sequences and structural organization of RNA segment 8 and the mRNAs coding for the NS1 and NS2 proteins.

Double-stranded DNA derived from influenza B virus genome RNA segment 8, which codes for the NS1 and NS2 proteins, was constructed by hybridization of full-length cDNA copies of RNA segment 8 and of the NS1 mRNA. This DNA was cloned in plasmid pBR322 and sequenced. The NS1 mRNA (approximately 1,080 viral nucleotides) contains nonviral nucleotides at its 5' end and is capable of coding for a protein of 281 amino acids. Sequencing of the NS2 mRNA has shown that it contains an interrupted sequence of 655 nucleotides and is most likely synthesized by a splicing mechanism. The first approximately 75 virus-specific nucleotides at the 5' end of the NS2 mRNA are the same as are found at the 5' -end of the NS1 mRNA. This region contains the initiation codon for protein synthesis and coding information for 10 amino acids common to the two proteins. The approximately 350-nucleotide body region of the NS2 mRNA can be translated in the +1 reading frame, and the sequence indicates that the NS1 and NS2 protein-coding regions overlap by 52 amino acids translated from different reading frames. Thus, between the influenza A and B viruses, the organization of the NS1 and NS2 mRNAs and the sizes of the NS2 mRNA and protein are conserved despite the larger size of the influenza B virus RNA segment, NS1 mRNA, and NS1 protein.     

The myelin-associated glycoproteins: membrane disposition, evidence of a novel disulfide linkage between immunoglobulin-like domains, and posttranslational palmitylation

The myelin-associated glycoproteins (MAG) are members of the immunoglobulin gene superfamily that function in the cell interactions of myelinating glial cells with axons. In this paper, we have characterized the structural features of these proteins. The disposition of MAG in the bilayer as a type 1 integral membrane protein (with an extracellularly disposed amino terminus, single transmembrane segment, and cytoplasmic carboxy terminus) was demonstrated in protease protection studies of MAG cotranslationally inserted into microsomes in vitro and in immunofluorescent studies with site specific antibodies. A genetically engineered MAG cDNA, which lacks the putative membrane spanning segment, was constructed and shown to encode a secreted protein. These results confirm the identify of this hydrophobic sequence as the transmembrane segment. Sequencing of the secreted protein demonstrated the presence of a cleaved signal sequence and the site of signal peptidase cleavage. To characterize the disulfide linkage pattern of the ectodomain, we cleaved MAG with cyanogen bromide and used a panel of antibodies to coprecipitate specific fragments under nonreducing conditions. These studies provide support for a novel disulfide linkage between two of the immunoglobulin domains of the extracellular segment. Finally, we report that MAG is posttranslationally palmitylated via an intramembranous thioester linkage. Based on these studies, we propose a model for the conformation of MAG, including its RGD sequence, which is considered with regard to its function as a cell adhesion molecule.     

Type XIV collagen is a variant of undulin.

We have isolated undulin, an extracellular matrix protein associated with the surface of collagen fibrils, from chicken embryos. The protein showed a molecular mass of about 600 kDa and is composed of three 210-kDa subunits linked by reducible as well as non-reducible bonds. In contrast to human undulin which reportedly is devoid of collagenous sequences, the chicken protein contained a short triple-helical segment that was sensitive to digestion by bacterial collagenase. Screening of an expression library with affinity-purified antibodies yielded two cDNA clones specific for chicken undulin. Analysis of the amino acid sequence deduced from the nucleotide sequence of these clones showed that the human and the chicken protein shared 71% sequence identity. At the amino-terminus both polypeptides contained several similar repeats related to the type III modules found in fibronectin. Towards the carboxyl terminus, however, the two sequences diverged substantially from each other. While the human sequence terminated in a proline-rich segment, the chicken sequence continued with a domain related to von Willebrand factor, with a domain similar to the noncollagenous domain NC4 of type IX collagen and with a typical collagenous triple helix. A short segment of this sequence was found to be identical with the published sequence of a bovine peptide derived from type XIV collagen. Our protein must therefore represent chicken type XIV collagen. One way to explain these results is the possibility that undulin exists in at least two alternatively spliced variants, one lacking the collagenous domain, as described initially for human undulin, and one containing the triple-helical domain, as found in type XIV collagen.     

水稻南方黑条矮缩病毒湖南鼎城株系S10片段的基因组序列分析

运用RT-PCR技术克隆了水稻南方黑条矮缩病毒(southern rice black-streaked dwarf virus,SRBSDV)湖南鼎城株系的基因组S10片段(SRBSDV-HuNDCS10),并对其全序列进行了测定和生物信息学分析。结果显示,SRBSDV-HuNDC S10片段全长为1797bp(登录号:JQ337964),含有1个ORF,编码557个氨基酸残基的衣壳蛋白,推测分子量约62.6kD,推测等电点为7.62,与已报道的广东、海南和云南分离物病毒的S10作比较,它们的核苷酸相似性分别为99.7%、99.0%和98.4%,氨基酸相似性分别为100.0%、99.5%和99.3%。对SRBSDV-HuNDCS10及部分Fijiviruses病毒对应片段在5’URT与3’URT存在的保守序列和互补序列进行了归纳,对其ORF编码的氨基酸序列进行了motif查找,得到该属(Fijiviruses)氨基酸序列的10个保守区段。此外,进行了糖基化位点、磷酸化位点及B细胞抗原表位预测,发现了3个可能的N端豆蔻酰基化位点,可能与病毒的侵染机制有关。