Homologous and unique G protein alpha subunits in the nematode Caenorhabditis elegans.

A cDNA corresponding to a known G protein alpha subunit, the alpha subunit of Go (Go alpha), was isolated and sequenced. The predicted amino acid sequence of C. elegans Go alpha is 80-87% identical to other Go alpha sequences. An mRNA that hybridizes to the C. elegans Go alpha cDNA can be detected on Northern blots. A C. elegans protein that crossreacts with antibovine Go alpha antibody can be detected on immunoblots. A cosmid clone containing the C. elegans Go alpha gene (goa-1) was isolated and mapped to chromosome I. The genomic fragments of three other C. elegans G protein alpha subunit genes (gpa-1, gpa-2, and gpa-3) have been isolated using the polymerase chain reaction. The corresponding cosmid clones were isolated and mapped to disperse locations on chromosome V. The sequences of two of the genes, gpa-1 and gpa-3, were determined. The predicted amino acid sequences of gpa-1 and gpa-3 are only 48% identical to each other. Therefore, they are likely to have distinct functions. In addition they are not homologous enough to G protein alpha subunits in other organisms to be classified. Thus C. elegans has G proteins that are identifiable homologues of mammalian G proteins as well as G proteins that appear to be unique to C. elegans. Study of identifiable G proteins in C. elegans may result in a further understanding of their function in other organisms, whereas study of the novel G proteins may provide an understanding of unique aspects of nematode physiology.     

Existence of two gamma subunits of the G proteins in brain

Although amino acid sequences have been determined for the alpha and beta subunits of Gs, Gi, and Go, sequences have not been reported for the gamma subunits of these G proteins. In the present paper, we determined the sequences of peptides prepared by partial proteolysis of two different forms of the gamma subunit of Gs, Gi, and Go from bovine brain. Using oligonucleotide probes based on the sequences of two of these peptides, a cDNA clone was isolated from a bovine adrenal cDNA library. This clone contained a 0.9-kilobase cDNA insert that included an open reading frame of 213 bases encoding a 71-amino acid polypeptide with an estimated Mr of 7850. The amino acid sequence predicted for the adrenal cDNA clone was identical to that determined for one form of the gamma subunit from brain. In addition, an antibody to a peptide based on the predicted amino acid sequence of this cDNA clone reacted specifically with one of the brain gamma subunits, indicating the adrenal cDNA clone encodes a gamma subunit present in both adrenal gland and brain. Also, evidence is presented, demonstrating the existence of a second, structurally distinct, form of the gamma subunit of Gs, Gi, and Go in brain.     

Characterization of a cDNA clone encoding a Brassica napus 12 S protein (cruciferin) subunit. Relationship between precursors and mature chains

Cruciferin (12 S globulin) is a large, neutral, oligometric protein synthesized in rapeseed (Brassica napus) during seed development. It is the major seed protein and is composed of six subunit pairs. Each of these pairs is synthesized as a precursor containing one heavy alpha-chain and one light beta-chain. Electrophoretic analysis of cruciferin showed that four different alpha- and four different beta-chains exist. A cruciferin clone was selected from an embryo cDNA library. This clone, pCRU1, contains a 1518-base pair open reading frame corresponding to a truncated NH2-terminal signal sequence followed by an alpha-chain of 296 and a beta-chain of 190 amino acid residues. Individual cruciferin chains as well as peptides thereof were subjected to NH2-terminal amino acid sequence analysis. The sequences obtained from a specific alpha- and beta-chain pair (alpha 1 and beta 1) showed total identity with the deduced amino acid sequence from pCRU1. Further comparisons revealed that a previously characterized cruciferin cDNA clone encodes one of the precursors for the closely related alpha 2/ alpha 3-beta 2/beta 3 subunits. The deduced amino acid sequences of the two cDNA clones display 64% similarity.     

The alpha- and beta-subunits of the human UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase [corrected] are encoded by a single cDNA

Lysosomal enzymes are targeted to the lysosome through binding to mannose 6-phosphate receptors because their glycans are modified with mannose 6-phosphate. This modification is catalyzed by UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase). Bovine GlcNAc-phosphotransferase was isolated using monoclonal antibody affinity chromatography, and an alpha2beta2gamma2-subunit structure was proposed. Although cDNA encoding the gamma-subunit has been described, cDNAs for the alpha- and beta-subunits have not. Using partial amino acid sequences from the bovine alpha- and beta-subunits, we have isolated a human cDNA that encodes both the alpha- and beta-subunits. Both subunits contain a single predicted membrane-spanning domain. The alpha- and beta-subunits appear to be generated by a proteolytic cleavage at the Lys928-Asp929 bond. Transfection of 293T cells with the alpha/beta-subunits-precursor cDNA with or without the gamma-subunit cDNA results in a 3.6- or 17-fold increase in GlcNAc-phosphotransferase activity in cell lysates, suggesting that the precursor cDNA contains the catalytic domain. The sequence lacks significant similarity with any described vertebrate enzyme except for two Notch-like repeats in the alpha-subunit. However, a 112-amino acid sequence is highly similar to a group of bacterial capsular polymerases (46% identity). A BAC clone containing the gene that spanned 85.3 kb and was composed of 21 exons was sequenced and localized to chromosome 12q23. We now report the cloning of both the cDNA and genomic DNA of the precursor of Glc-NAc-phosphotransferase. The completion of cloning all three subunits of GlcNAc-phosphotransferase allows expression of recombinant enzyme and dissection of lysosomal targeting disorders.     

Rat clusterin isolated from primary Sertoli cell-enriched culture medium is sulfated glycoprotein-2 (SGP-2)

Clusterin, a glycoprotein originally isolated from ram rete testis fluid, is a dimer composed of monomers with non-identical NH2-terminal amino acid sequences. In view of its possible role in cell-cell interactions in the seminiferous epithelium, we sought to identify such a protein in the rat. Using the bioassay developed for the ovine protein, rat clusterin was purified to apparent homogeneity by HPLC from primary Sertoli cell-enriched culture media. This protein is also a heterodimer consisting of monomers of Mr 43,000 (alpha) and Mr 35,000 (beta). NH2-Terminal amino acid sequence analysis indicated that the alpha subunit has a sequence of NH2-SLMPLSHYGPLSFHNMFQPFFDMIHQAQQA and the beta subunit, NH2-EQEFSDNELQELSTQGSRYVNKEIQNAVQG. These two subunits show marked similarity with the corresponding subunits of ram clusterin isolated from rete testis fluid. Using an antibody against the alpha subunit of rat clusterin, a cDNA clone was isolated from a rat testicular lambda gt11 cDNA library. Analyses of the amino acid sequence derived from the isolated rat clusterin cDNA and of the NH2-terminal amino acid sequences indicate that rat clusterin is identical to a Sertoli cell glycoprotein previously designated sulfated glycoprotein-2.     

Identification of three pertussis toxin substrates (41, 40 and 39 kDa proteins) in mammalian brain. Comparison of predicted amino acid sequences from G-protein alpha-subunit genes and cDNAs with partial amino acid sequences from purified proteins

We have determined the partial amino acid sequences of the 40 kDa protein, one of the three pertussis toxin substrates in porcine brain. Purified 40 kDa protein from porcine brain was completely digested with TPCK-trypsin. Digested peptides were separated by reverse-phase HPLC and subjected to analysis by gas-phase protein sequencing. Several sequences of porcine brain 40 kDa protein completely matched with those which were deduced from the nucleotide sequences of the human Gi2 alpha gene and rat Gi2 alpha cDNA. On the other hand, the previously determined sequences of the rat brain 41 and 39 kDa proteins were in complete agreement with the predicted amino acid sequences of rat Gi1 alpha and Go alpha cDNAs, respectively.     

Drosophila contains three genes that encode distinct isoforms of protein phosphatase 1

The sequences of two Drosophila and one rabbit protein phosphatase (PP) 1 catalytic subunits were determined from their cDNA. The sequence of Drosophila PP1 alpha 1 was deduced from a 2.2-kb cDNA purified from an embryonic cDNA library, while that for Drosophila PP1 beta was obtained from overlapping clones isolated from both a head cDNA library and an eye imaginal disc cDNA library. The gene for Drosophila PP1 alpha 1 is at 96A2-5 on chromosome 3 and encodes a protein of 327 amino acids with a calculated molecular mass of 37.3 kDa. The gene for Drosophila PP1 beta is localized at 9C1-2 on the X chromosome and encodes a protein of 330 amino acids with a predicted molecular mass of 37.8 kDa. PP1 alpha 1 shows 96% amino acid sequence identity to PP1 alpha 2 (302 amino acids), an isoform whose gene is located in the 87B6-12 region of chromosome 3 [Dombrádi, V., Axton, J. M., Glover, D.M. Cohen, P.T.W. (1989) Eur. J. Biochem. 183, 603-610]. PP1 beta shows 85% identity to PP1 alpha 1 and PP1 alpha 2 over the 302 homologous amino acids. These results demonstrate that at least three genes are present in Drosophila that encode different isoforms of PP1. Drosophila PP1 alpha 1 and PP1 beta show 89% amino acid sequence identity to rabbit PP1 alpha (330 amino acids) [Cohen, P.T.W. (1988) FEBS Lett. 232, 17-23] and PP1 beta (327 amino acids), respectively, demonstrating that the structures of both isoforms are among the most conserved proteins known throughout the evolution of the animal kingdom. The presence of characteristic structural differences between PP1 alpha and PP1 beta, which have been preserved from insects to mammals, implies that the alpha and beta isoforms may have distinct biological functions.     

Characterization of the subunits of beta-conglycinin

Four subunits of beta-conglycinin were purified from soybean cultivar CX 635-1-1-1, and were designated alpha, alpha', beta, and beta' in accordance with nomenclature proposed by Thanh and Shibasaki [(1977) Biochim. Biophys. Acta 490, 370-384]. Of these subunits, beta' has not previously been reported or characterized. Consistent with the low levels of methionine in these proteins, cyanogen bromide cleavage of alpha', alpha, and beta' subunits produced only a few fragments. The beta subunit contains no methionine and was not cleaved by cyanogen bromide. The NH2-terminal amino acid sequences of the alpha and alpha' subunits are homologous, and each has valine at its amino terminus. The beta subunit has a very different NH2-terminal sequence from those of the alpha and alpha' subunits, and has leucine at its amino terminus. The NH2-terminal sequence of the beta' subunit could not be determined, as it appeared to be blocked to Edman degradation. Although alpha and alpha' subunits have similar NH2-terminal sequences, they differ in the number of methionine residues and so yielded different numbers of cyanogen bromide fragments. Two cyanogen bromide fragments (CB-1 and CB-2) were purified from the alpha subunit. CB-1 originated from the NH2-terminal end of the subunit. The amino acid sequence of CB-2 was identical to that predicted from the nucleotide sequence of cDNA clone pB36. The insert in pB36 encoded 216 amino acids from the COOH-terminal end of the alpha subunit and contained a 138-bp trailer sequence which was followed by a poly-(A) tail. Maps showing the relative positions of methionine residues and carbohydrate moieties in the alpha and alpha' subunits were drawn, based on primary sequence data, and the size and carbohydrate content of the CNBr fragments derived from the subunits.     

Cloning and expression of Drosophila melanogaster UDP-GlcNAc:alpha-3-D-mannoside beta1,2-N-acetylglucosaminyltransferase I

A TBLASTN search of the Drosophila melanogaster expressed sequence tag (EST) database with the amino acid sequence of human UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT I, EC 2.4.1.101) as probe yielded a clone (GM01211) with 56% identity over 36 carboxy-terminal amino acids. A 550 base pair (bp) probe derived from the EST clone was used to screen a Drosophila cDNA library in lambda-ZAP II and two cDNAs lacking a start ATG codon were obtained. 5'-Rapid amplification of cDNA ends (5'-RACE) yielded a 2828 bp cDNA containing a full-length 1368 bp open reading frame encoding a 456 amino acid protein with putative N-terminal cytoplasmic (5 residues) and hydrophobic transmembrane (20 residues) domains. The protein showed 52% amino acid sequence identity to human GnT I. This cDNA, truncated to remove the N-terminal hydrophobic domain, was expressed in the baculovirus/Sf9 system as a secreted protein containing an N-terminal (His)6 tag. Protein purified by adsorption to and elution from nickel beads converted Man alpha1-6(Man alpha1-3)Man beta-octyl (M3-octyl) to Man alpha1-6(GlcNAc beta1-2Man alpha1-3)Man beta-octyl. The Km values (0.7 and 0.03 mM for M3-octyl and UDP-GlcNAc respectively), temperature optimum (37 degrees C), pH optimum (pH 5 to 6) and divalent cation requirements (Mn > Fe, Mg, Ni > Ba, Ca, Cd, Cu) were similar to mammalian GnT I. TBLASTN searches of the Berkeley Drosophila Genome Project database with the Drosophila GnT I cDNA sequence as probe allowed localization of the gene to chromosomal region 2R; 57A9. Comparison of the cDNA and genomic DNA sequences allowed the assignment of seven exons and six introns; all introns showed GT-AG splice site consensus sequences. This is the first insect GnT I gene to be cloned and expressed.     

Complete nucleotide and derived amino acid sequences of the fourth component of mouse complement (C4). Evolutionary aspects

The nucleotide sequence coding for the fourth component of mouse complement (C4) has been determined from a cloned genomic DNA fragment and a cloned cDNA fragment. The amino acid sequence of the protein was deduced. The single chain precursor protein (pro-C4) consists of 1719 amino acid residues. The mature beta, alpha, and gamma subunits contain 654, 766, and 291 amino acids, respectively. One potential carbohydrate attachment site is predicted for the beta chain, three for the alpha chain, and none for the gamma chain. From a comparison with human C4 cDNA sequence an extensive overall sequence homology, 79% in nucleotides and 76% in amino acids, is observed. There is conservation in both the position and number of cysteine residues in human and mouse C4. We compared the mouse C4 amino acid sequences with those of mouse C3 and human alpha 2-macroglobulin and the evolutionary relationship among these three proteins is discussed.     

Alpha i-3 cDNA encodes the alpha subunit of Gk, the stimulatory G protein of receptor-regulated K+ channels

cDNA cloning has identified the presence in the human genome of three genes encoding alpha subunits of pertussis toxin substrates, generically called "Gi." They are named alpha i-1, alpha i-2 and alpha i-3. However, none of these genes has been functionally identified with any of the alpha subunits of several possible G proteins, including pertussis toxin-sensitive Gp's, stimulatory to phospholipase C or A2, Gi, inhibitory to adenylyl cyclase, or Gk, stimulatory to a type of K+ channels. We now report the nucleotide sequence and the complete predicted amino acid sequence of human liver alpha i-3 and the partial amino acid sequence of proteolytic fragments of the alpha subunit of human erythrocyte Gk. The amino acid sequence of the proteolytic fragment is uniquely encoded by the cDNA of alpha i-3, thus identifying it as alpha k. The probable identity of alpha i-1 with alpha p and possible roles for alpha i-2, as well as additional roles for alpha i-1 and alpha i-3 (alpha k) are discussed.     

The primary sequence and the subunit structure of mouse alpha-2-macroglobulin, deduced from protein sequencing of the isolated subunits and from molecular cloning of the cDNA.

Mouse plasma alpha-2-macroglobulin (m alpha 2M) was isolated and the N-terminal amino-acid sequences determined after separation of the 165-kDa and 35-kDa subunits. These sequences were compared to the protein sequence predicted by the cDNA, which was cloned from a mouse liver library and sequenced. From these data it is evident that both subunits are encoded by one mRNA of approximately 5 kb expressed predominantly in liver. The smaller subunit, with the N-terminal sequence DLSSSDLT, comprises the C-terminal 257 residues of m alpha 2M and is derived from a single-chain precursor probably by proteolytic processing at an arginine residue in the sequence PTRDLSS. Analysis of the predicted protein further showed all the salient features of a proteinase inhibitor of the macroglobulin family: a bait region that deviates from all known sequences in this family, a very conserved internal thiolester site and conserved cysteine residues and putative N-glycosylation sites. The synthesis of m alpha 2M in adult liver was demonstrated by Northern blotting and in fetal liver by in-situ hybridization. Transient transfection of COS cells with the cDNA under control of a viral promoter demonstrated the secretion and partial processing of m alpha 2M in the culture medium. In plasma the level of m alpha 2M was found to be stable as expected for the murine counterpart of human plasma alpha-2-macroglobulin. The possibilities of using the mouse as a genetic model to study this proteinase inhibitor in vivo are discussed.     

Construction and characterization of cDNA encoding the alpha 2 chain of chicken type IX collagen

We have isolated and characterized a cDNA encoding the carboxy-terminal half of one of the polypeptide subunits of a novel disulfide-bonded collagen found in hyaline cartilage. This collagen has been given the type assignment type IX, and it has several unusual characteristics. First, the polypeptide subunits are shorter than alpha-chains of the fibrillar collagens types I, II, and III. Second, type IX molecules are heterotrimers of three genetically distinct polypeptide subunits. Third, type IX molecules contain three triple-helical collagenous domains interspersed with noncollagenous domains. When chicken cartilage collagens are extracted with pepsin, type IX collagen is cleaved and gives rise to the triple-helical fragments HMW and LMW. The identification of the cDNA reported here is based on a comparison of the amino acid composition of tryptic peptides derived from LMW with the composition of tryptic peptides predicted from the nucleotide sequence of the cDNA. We also show that the amino-terminal sequence of one of the subunits of LMW is identical with the sequence predicted from the nucleotide sequence of the cDNA. Finally, we demonstrate that the amino-terminal amino acid sequence of a tryptic peptide isolated from one of the subunits of HMW is identical with a sequence predicted from the cDNA. We have given the polypeptide chain encoded by the cDNA reported here the name alpha 2(IX), and we show that it is homologous to the alpha 1(IX) chain previously characterized by us.     

Molecular cloning and sequence analysis of two rat major globin cDNAs

Two cDNA clones for globins of the adult Wistar rat were isolated from a reticulocyte cDNA library and the nucleotide sequences of the inserts were determined. One clone contained a cDNA insert consisting of 556 bp and the other contained one of 577 bp, both covering the entire coding sequences for rat globins. Comparisons of their predicted amino acid sequences with known sequences of rat globins revealed that these cDNAs coded for a rat major alpha- and a major beta-globin, I alpha and II beta, respectively. The cause of diversity of rat globins was discussed in terms of the nucleotide sequences of cDNAs and known amino acid sequences of globins.     

Structural requirements for efficient processing and activation of recombinant human UDP-N-acetylglucosamine:lysosomal-enzyme-N-acetylglucosamine-1-phosphotransferase

Mannose 6-phosphate-modified N-glycans are the determinant for intracellular targeting of newly synthesized lysosomal hydrolases to the lysosome. The enzyme responsible for the initial step in the synthesis of mannose 6-phosphate is UDP-N-acetylglucosamine:lysosomal-enzyme-N-acetylglucosmine-1-phosphotransferase(GlcNAc-phosphotransferase). GlcNAc-phosphotransferase is a multisubunit enzyme with an alpha2beta2gamma2 arrangement that requires a detergent for solubilization. Recent cloning of cDNAs and genes encoding these subunits revealed that the alpha- and beta-subunits are encoded by a single gene as a precursor, whereas the gamma-subunit is encoded by a second gene. The hydropathy plots of the deduced amino acid sequences suggested that the alpha- and beta-subunits but not the gamma-subunit contain transmembrane domains. Access to these cDNAs allowed us to express a soluble form of human recombinant GlcNAc-phosphotransferase by removing the putative transmembrane and cytoplasmic domains from the alpha- and beta-subunits. Because this modification prevented precursor processing to mature alpha- and beta-subunits, the native cleavage sequence was replaced by a cleavage site for furin. When the modified alpha/beta-subunits (alpha'/beta'-subunits) precursor and wild type gamma-subunit cDNAs were co-expressed in 293T or CHO-K1 cells, a furin-like protease activity in these cells cleaved the precursor and produced an active and processed soluble GlcNAc-phosphotransferase with an alpha'2beta'2gamma2-subunits arrangement. Recombinant soluble GlcNAc-phosphotransferase exhibited specific activity and substrate preferences similar to the wild type bovine GlcNAc-phosphotransferase and was able to phosphorylate a lysosomal hydrolase, acid alpha-glucosidase in vitro.     

Isolation and sequencing of cDNA clones encoding alpha and beta subunits of Drosophila melanogaster casein kinase II.

Cloned cDNAs encoding both subunits of Drosophila melanogaster casein kinase II have been isolated by immunological screening of lambda gt11 expression libraries, and the complete amino acid sequence of both polypeptides has been deduced by DNA sequencing. The alpha cDNA contained an open reading frame of 336 amino acid residues, yielding a predicted molecular weight for the alpha polypeptide of 39,833. The alpha sequence contained the expected semi-invariant residues present in the catalytic domain of previously sequenced protein kinases, confirming that it is the catalytic subunit of the enzyme. Pairwise homology comparisons between the alpha sequence and the sequences of a variety of vertebrate protein kinase suggested that casein kinase II is a distantly related member of the protein kinase family. The beta subunit was derived from an open reading frame of 215 amino acid residues and was predicted to have a molecular weight of 24,700. The beta subunit exhibited no extensive homology to other proteins whose sequences are currently known.     

Galactose-binding lectin from the seeds of champedak (Artocarpus integer): sequences of its subunits and interactions with human serum O-glycosylated glycoproteins

Our group has previously reported the isolation, partial characterisation, and application of a Galbeta1-3GalNAc- and IgA1-reactive lectin from the seeds of champedak (Artocarpus integer). In the present study, we have subjected the purified lectin to reverse-phase high performance liquid chromatography and sequenced its subunits. Determination of the N-terminal sequence of the first 47 residues of the large subunit demonstrated at least 95% homology to the N-terminal sequence of the alpha chains of a few other galactose-binding Artocarpus lectins. The two smaller subunits of the lectin, each comprised of 21 amino acid residues, demonstrated minor sequence variability. Their sequences were generally comparable to the beta chains of the other galactose-binding Artocarpus lectins. When used to probe human serum glycopeptides that were separated by two-dimensional gel electrophoresis, the lectin demonstrated strong apparent interactions with glycopeptides of IgA1, hemopexin, alpha2-HS glycoprotein, alpha1-antichymotrypsin, and a few unknown glycoproteins. Immobilisation of the lectin to Sepharose generated an affinity column that may be used to isolate the O-glycosylated serum glycoproteins.