An islet-cell protein tyrosine phosphatase is a likely precursor to the 37-kDa autoantigen in type 1 diabetes: human and macaque sequences, tissue distribution, unique and shared epitopes, and predictive autoantibodies.

BACKGROUND: We sought to identify novel islet-cell autoantigens to better understand the pathogenesis, prediction, and immunotherapy of type 1 diabetes. MATERIALS AND METHODS: Macaque and human islet cDNA libraries expressed in mammalian cells were screened with human diabetes sera. A positive clone was sequenced directly and after 5' rapid amplification of cDNA ends (RACE). Northern blotting and in situ hybridization revealed the tissue distribution of the corresponding protein. Antigen, expressed by in vitro translation, and tryptic peptides were analyzed by SDS-PAGE. For the immunoprecipitations, 183 diabetic, 60 prediabetic, and 91 control sera were used. Truncated antigens were used in immunoprecipitations for epitope mapping. Recombinant antigen expressed in transfected fibroblasts was used in competition assays. RESULTS: Sequencing yielded an 111-kDa, 1,013 amino acid, transmembrane protein (M1851) containing consensus protein tyrosine phosphatase (PTPase) sequence. M1851 was 77% identical in the intracellular domain, but only 31% identical extracellularly, to the islet-cell autoantigen ICA512. mRNA localized to brain, prostate, pancreatic islets, and adrenal medulla. After limited trypsinization, the in vitro translated antigen was 37 kDa. M1851 was recognized by 47% of prediabetes sera, 31% of new diabetes sera, but only 1% of healthy controls. Only 1/73 sera binding M1851 failed to bind ICA512, whereas 42/114 binding ICA512 did not bind M1851. M1851 reactivity was not fully displaced by ICA512 in 24/49 sera. Removing the C-terminal 27, 80, or 160 amino acids of M1851 decreased reactivity by 70%, 90%, and 100%, respectively. CONCLUSIONS: This new islet-cell PTPase is likely to be the precursor to the 37-kDa tryptic fragment antigen. It is structurally related to ICA512 but has distinct diabetes autoantibody epitopes located at the C terminus.     

Molecular cloning and sequence analysis of full-length cDNA for mRNA of adrenodoxin oxidoreductase from bovine adrenal cortex

A full-length cDNA clone (pADR) for adrenodoxin reductase was isolated by means of immunological screening from a bovine adrenal poly(A)+ mRNA library. A cDNA insert of 1,973 base pairs in length encoded the entire amino acid sequence of the adrenodoxin reductase precursor protein, which consists of 492 amino acids including an extrapeptide of 32 amino acids at the NH2-terminus. The cloned cDNA contained the complete 3'-noncoding region of 443 nucleotides including 59 nucleotides of poly(A) and 51 nucleotides in the 5'-noncoding region. The amino acid sequences from the 33rd to 70th, the 117th to 123rd, the 207th to 225th, the 247th to 323rd, the 385th to 426th, the 444th to 461st, and the 487th to 492nd in the predicted structure were identical with those of the purified adrenodoxin reductase and its digested peptides, with only four exceptions.     

野生大豆(Glycine soja Sieb et ZUCC)球蛋白A_5 A_4 B_3 cDNA的研究(简报)

大豆球蛋白是大豆种子中主要的贮藏蛋白。它们在某些作物中占种子干重20%以上。已经知道大豆球蛋白由6个亚基组成。每个亚基由一或两个酸性多肽(A)和一个碱性多肽(B)组成,多肽之间由二硫键联结。这些亚基是从编码A-B亚基前体的mRNA合成,然后经过转录后加工剪切形成A肽和B肽。至今所有关于球蛋白的基因结构与表达的报道都集中于栽培大豆上。由于我国有丰富的大豆     

Cloning of the human cDNA for the U1 RNA-associated 70K protein.

Anti-RNP sera were used to isolate a cDNA clone for the largest polypeptide of the U1 snRNP, a protein of mol. wt 70 kd designated 70K, from a human liver cDNA library constructed in the expression vector pEX1. The cro-beta-galactosidase-70K fusion protein reacted with various anti-RNP patient sera, a rabbit anti-70K antiserum, as well as with a monoclonal antibody specific for this protein. The sequences of four 70K peptides were determined and they match parts of the deduced amino acid sequence of the 1.3 kb insert of p70.1 indicating that it is a genuine 70K cDNA. Screening of a new cDNA library constructed from polysomal mRNA of HeLa cells with the p70.1 clone yielded an overlapping clone, FL70K, which was 2.7 kb long and covered the complete coding and 3'-untranslated sequence of the 70K protein in addition to 680 nucleotides upstream of the putative initiation codon, The predicted mol. wt of the encoded protein is approximately 70 kd. Amino acid analysis of the purified HeLa 70K protein yielded values close or identical to those deduced from the nucleotide sequence of the full-length cDNA. The 70K protein is rich in arginine (20%) and acidic amino acids (18%). Extremely hydrophilic regions containing mixed-charge amino acid clusters have been identified at the carboxyl-terminal half of the protein, which may function in RNA binding. A sequence comparison with two recently cloned RNA binding proteins revealed homology with one region in the U1 RNP 70K protein. This domain may also be responsible for RNA binding.     

Nucleotide sequence encoding the precursor of the small subunit of ribulose 1,5-bisphosphate carboxylase from Lemna gibba L.G-3

We have sequenced a cDNA clone, pLgSSU, which encodes the small subunit of ribulose 1,5-bisphosphate carboxylase of Lemna gibba L.G-3 a monocot plant. This clone contains a 832 basepair insert which encodes the entire 120 amino acids of the mature small subunit polypeptide (Mr = 14,127). In addition this clone encodes 53 amino acids of the amino terminal transit peptide of the precursor polypeptide and 242 nucleotides of the 3' non-coding region. Comparison of the nucleotide sequence of pLgSSU with Lemna gibba genomic sequences homologous to the 5' end of the cDNA clone suggests that nucleotides encoding four amino-terminal amino acids of the transit peptide are not included in the cDNA clone. The deduced amino acid sequence of the Lemna gibba mature small subunit polypeptide shows 70-75% homology to the reported sequences of other species. The transit peptide amino acid sequence shows less homology to other species. There is 50% homology to the reported soybean sequence and only 25% homology to the transit sequence of another monocot, wheat.     

The nucleotide sequence and derived amino acid sequence of cDNA coding for mouse carbonic anhydrase II

The nucleotide sequence of a clone containing mouse carbonic anhydrase (CA) cDNA in pBR322 has been determined. The cloned cDNA contains all of the coding region except for nucleotides specifying the first eight amino acids, and all of the 3' noncoding region, which consists of 700 nucleotides. A cDNA clone was identified which contains an additional 54 bp at the 5' end, so that the complete amino acid sequence of mouse CA could be deduced. This sequence showed a 73-81% homology with other mammalian CA form II isozymes, 56-63% with form I isozymes, and 52-56% with form III isozymes. By examination of the amino acids which are unique and invariant for each isozyme, the mouse amino acid sequence was found to contain 16 of the 23 residues that are unique and invariant to mammalian CA form II isozymes, but only one or no residue for forms I and III, respectively.     

Nucleotide sequence of the cDNA and the derived amino acid sequence for the major antigenic protein of foot and mouth disease virus, type Asia 1 63/72.

A 0.9 kb cDNA for the foot and mouth disease virus (FMDV) type Asia 1 63/72, cloned in the plasmid pUR222 by dC/dG tailing method, was expressed into a protein which was immunogenic in guinea pigs and cattle. The protein purified to homogeneity was found to be basic and of 38 kDa. A sequence of 879 nucleotides of the inserted cDNA was obtained. The nucleotide sequence was 65% GC-rich and was homologous to the gene for VPI of FMDV types A5, OIK and C3 to the extent of 35-40%. From the nucleotide sequence, a sequence of 293 amino acids was derived which contained 43 arginine, 4 lysine, 7 glutamic acid and 18 aspartic acid residues making the protein highly basic. The molecular weight was calculated to be 31.6 kDa. The 38 kDa protein produced by the cloned cDNA is a fused protein composed of the 293 amino acids; 5 and 55 amino acids of the alpha-complementation protein of the beta-galactosidase at the N and C terminal, respectively, and 5 amino acid coded by the dG/dC tails used for cloning the cDNA.     

Structural requirement for autoreactivity on human pyruvate dehydrogenase-E2, the major autoantigen of primary biliary cirrhosis. Implication for a conformational autoepitope.

The E2 component (acetyltransferase) of the pyruvate dehydrogenase (PDH) complex is the major mitochondrial autoantigen recognized by autoantibodies in patients with primary biliary cirrhosis (PBC). Previous work, using only a partial length rat liver cDNA clone of PDH-E2, demonstrated that the immunodominant epitope was localized to the lipoic acid binding site. Human PDH-E2, in contrast to rat PDH-E2, has two lipoic acid binding sites. By using a full length human cDNA for PDH-E2, and by preparation of multiple overlapping recombinant fragments, we have determined that three autoreactive determinants are present on human PDH-E2: two cross-reactive lipoyl domains, and an area surrounding the E1/E3 binding region. The dominant epitope was localized to the inner lipoyl domain whereas the outer lipoyl domain only showed a weak cross-reactivity, and only 1/26 PBC sera reacted weakly to the E1/E3 binding region area. By probing recombinant fusion proteins expressed from small restriction fragments of the inner lipoyl domain, we have found that a minimum of 75 amino acids (residues 146-221) were required for detectable autoantibody binding, and that 93 amino acids (residues 128-221) were necessary for characteristically strong antimitochondrial autoantibody recognition. Such a requirement for a large region suggests the possibility that a conformational autoepitope may be recognized. In addition, we have found that absorption of PBC sera with the purified mammalian PDH complex does not remove reactivity against Escherichia coli Ag. The possible implications for such results are discussed.     

Rat liver glutathione S-transferases. Nucleotide sequence analysis of a Yb1 cDNA clone and prediction of the complete amino acid sequence of the Yb1 subunit

We have constructed a nearly full length cDNA clone, pGTA/C44, complementary to the rat liver glutathione S-transferase Yb1 mRNA. The nucleotide sequence of pGTA/C44 has been determined, and the complete amino acid sequence of the Yb1 subunit has been deduced. The cDNA clone contains an open reading frame of 654 nucleotides encoding a polypeptide comprising 218 amino acids with Mr = 25,919. The NH2-terminal sequence deduced from DNA sequence analysis of pGTA/C44 is in agreement with the first 19 amino acids determined for purified glutathione S-transferase A, a Yb1 homodimer, by Frey et al. (Frey, A. B., Friedberg, T., Oesch, F., and Kreibich, G. (1983) J. Biol. Chem. 258, 11321-11325). The DNA sequence of pGTA/C44 shares significant sequence homology with a cDNA clone, pGT55, which is complementary to a mouse liver glutathione S-transferase (Pearson, W. R., Windle, J. J., Morrow, J. F., Benson, A. M., and Talalay, P. (1983) J. Biol. Chem. 258, 2052-2062). We have also determined 37 nucleotides of the 5'-untranslated region and 348 nucleotides of the 3'-untranslated region of the Yb1 mRNA. The Yb1 mRNA and subunit do not share any sequence homology with the rat liver glutathione S-transferase Ya or Yc mRNAs or their corresponding subunits. These data provide the first direct evidence that the Yb1 subunit is derived from a gene or gene family which is distinct from the Ya-Yc gene family.     

Cloning and expression of a 12 kDa serospecific epitope of Wuchereria bancrofti

The immunoscreening of a microfilarial cDNA library of Wuchereria bancrofti with microfilaraemic sera revealed many positive clones expressing filarial antigens. One immunoreactive clone, designated PMR1, was shown to encode a protein of 114 amino acid residues. The cDNA fragment was subcloned into an expression vector, Pinpoint XaT. The resulting recombinant (r)PMR1-biotin fusion protein was expressed in Escherichia coli (BL21 [DE3] pLys) and was affinity purified on avidin resin. Analysis of sera of different groups for filarial antibodies against rPMR1 showed it to be highly reactive with microfilaraemic and clinical filarial sera compared to its reactivity with endemic and nonendemic controls. This indicates that the gene sequence of cDNA is expressing an immunodominant epitope, which could be useful in serodiagnosis of lymphatic filariasis.     

Molecular cloning and functional identification of a ribosome inactivating/antiviral protein from leaves of post-flowering stage of Celosia cristata and its expression in E. coli

A full-length cDNA clone, encoding a ribosome inactivating/antiviral protein (RIP/AVP) was isolated from the cDNA library of post-flowering stage of Celosia cristata leaves. The full-length cDNA consisted of 1015 nucleotides, with an open reading frame encoding 283 amino acids. The deduced amino acid sequence had a putative active site domain conserved in other ribosome inactivating/antiviral proteins (RIPs/AVPs). The coding region of the cDNA was amplified by polymerase chain reaction (PCR), cloned and expressed in Escherichia coli as recombinant protein of 72 kDa. The expressed fusion product was confirmed by Western analysis and purification by affinity chromatography. Both the recombinant protein (reCCP-27) and purified expressed protein (eCCP-27) inhibited translation in rabbit reticulocytes showing IC50 values at 95 ng and 45 ng, respectively. The native purified nCCP-27 has IC50 at 25 ng. The purified product also showed N-glycosidase activity towards tobacco ribosomes and antiviral activity towards tobacco mosaic virus (TMV) and sunnhemp rosette virus (SRV).     

Purification, characterization and cloning of antiviral/ribosome inactivating protein from Amaranthus tricolor leaves

An antiviral protein (AVP), imparting high level of resistance against sunnhemp rosette virus (SRV) was purified from the dried leaves of Amaranthus tricolor. The purified protein (AAP-27) exhibited approximately 98% inhibition of local lesion formation at a concentration range of approximately 30 microg ml(-1). The protein was found to be highly basic glycoprotein monomer (pI approximately 9.8) of Mr 27 kDa, with neutral sugar content of 4%. The purified protein exhibited N-glycosidase and RNase activities. We have also isolated full-length cDNA clone, encoding this protein designated as A. tricolor antiviral protein-1 (AAP-1). Two primers, one designed on the basis of N-terminal sequence of the purified protein and the other from the conserved active peptides of other AVPs/RIPs were used for PCR amplification of double stranded cDNA, isolated from the leaves of A. tricolor. The amplified fragment was used as a probe for library screening. The isolated full-length cDNA consisted of 1058 nucleotides with an open reading frame encoding a polypeptide of 297 amino acids. The deduced amino acid sequence of AAP-1 has a putative active domain conserved in other AVPs/RIPs and shows varying homology to the RIPs from other plant species.     

Isolation and sequence analysis of a complementary DNA encoding rat liver L-gulono-gamma-lactone oxidase, a key enzyme for L-ascorbic acid biosynthesis

L-Gulono-gamma-lactone oxidase, one of the microsomal flavin enzymes, catalyzes the last step of L-ascorbic acid biosynthesis in many animals; however, it is missing in scurvy-prone animals such as humans, primates, and guinea pigs. A cDNA clone for this enzyme was isolated by screening a rat liver cDNA expression library in lambda gt11 using antibody directed against the enzyme. The cDNA clone contained 2120 nucleotides and an open reading frame of 1320 nucleotides encoding 440 amino acids of the protein with a molecular weight of 50,605. The amino-terminal sequence (residues 1-33) of the enzyme isolated from rat liver completely coincided with the corresponding part of the deduced amino acid sequence. The identity of the cDNA clone was further confirmed by the agreement of the composition of the deduced amino acids with that determined by amino acid analysis of the enzyme. Hydropathy analysis of the deduced amino acid sequence revealed several hydrophobic regions, suggesting that they anchor the protein into the microsomal membrane. The deduced amino acid sequence showed no obvious homology with the flavin-binding regions of other eight flavoenzymes.     

A cDNA clone encoding a glycinin A1a subunit precursor of soybean.

A cDNA clone covering the whole coding region for a glycinin subunit precursor containing the A1a acidic subunit, one of the A2 family, has been identified from a library of soybean cotyledonary cDNA clones using a mixed oligonucleotide probe. Analysis of the cDNA insert revealed that it contained 1746 nucleotides of mRNA sequence with a 5'-terminal nontranslated region of 54 nucleotides, a signal peptide region corresponding to 19 amino acids, an acidic subunit region (A1a) corresponding to 291 amino acids followed by a basic subunit region corresponding to 185 amino acids, and a 3'-terminal nontranslated region of 207 nucleotides. By comparing the predicted protein sequence of this precursor with that of the legumin A precursor of pea, it was found that glycinin A2 subunit family appeared to be more closely related to the legumin than to the A3 subunit family, and that the evolutional rearrangement of glycinin genes has occurred.     

Cloning of a cDNA encoding a new ribosome-inactivating protein from Beta vulgaris vulgaris (mangold)

By means of a λZAP II cDNA library constructed from seedlings of Beta vulgaris vulgaris and immunoscreening, a cDNA clone containing a partial sequence of a new ribosome-inactivating protein (RIP) was obtained. As confirmed by Western blot analysis, this clone produced a RIP upon induction with IPTG. We called it betavulgin (Bvg). The recombinant protein (re-Bvg) was somewhat smaller than plant-derived RIP (28 versus 30 and 32 kDa), but showed the specific N-glycosidase activity on tobacco ribosomes, confirming its RIP character. The cDNA was sequenced and the missing 5'-end was established by RACE using bvg-specific primers. The entire cDNA was 1080 nucleotides in length and encoded a protein of 272 amino acids with a sequence identity of 26–40% with other RIP.