Structure and evolution of the lipase superfamily.

The lipase superfamily includes three vertebrate and three invertebrate (dipteran) proteins that show significant amino acid sequence similarity to one another. The vertebrate proteins are lipoprotein lipase (LPL), hepatic lipase (HL), and pancreatic lipase (PL). The dipteran proteins are Drosophila yolk proteins 1, 2, and 3. We review the relationships among these proteins that have been established according to gene structural relatedness and introduce our findings on the phylogenetic relationships, distance relationships, and evolutionary history of the lipase gene superfamily. Drosophila yolk proteins contain a 104 amino acid residue segment that is conserved with respect to the lipases. We have used the yolk proteins as an outgroup to root a phylogeny of the lipase family. Our phylogenetic reconstruction suggests that ancestral PL diverged earlier than HL and LPL, which share a more recent root. Human and bovine LPL are shown to be more closely related to murine LPL than to guinea pig LPL. A comparison of the distance (a measure of the number of substitutions between sequences) between mammalian and avian LPL reveals that guinea pig LPL has the largest distance from the other mammals. Human, rodent, and rabbit HL show marked divergence from one another, although they have similar relative rates of amino acid substitution when compared to human LPL as an outgroup. Human and porcine PL are not as divergent as human and rat HL, suggesting that PL is more conserved than HL. However, canine PL demonstrates an unusually rapid rate of substitution with respect to the other pancreatic lipases. The lipases share several structurally conserved features. One highly conserved sequence (Gly-Xaa-Ser-Xaa-Gly) contains the active site serine. This feature, which agrees with that found in serine esterases and proteases, is found within the entire spectrum of lipases, including the evolutionarily unrelated prokaryotic lipases. We review the location and possible activity of putative lipid binding domains. We have constructed a conservation index (CI) to display conserved structural features within the lipase gene family, a CI of 1.0 signifying perfect conservation. We have found a correlation between a high CI and the position of conserved functional structures. The putative lipid-binding domains of LPL and HL, the disulfide-bridging cysteine residues, catalytic residues, and N-linked glycosylation sites of LPL, HL, and PL all lie within regions having a CI of 0.8 or higher. A number of amino acid substitutions have been identified in familial hyperchylomicronemia which result in loss of LPL function.(ABSTRACT TRUNCATED AT 400 WORDS)     

A High Mobility Group Protein from the Dipteran Insects Ceratitis capitata and Bactrocera oleae

Nuclei from Bactrocera oleae and Ceratitis capitata larvae contain a major protein that shares most of the characteristics of vertebrate high mobility group (HMG) proteins. Proteins are extracted from nuclei with 0.35 M NaCl, are soluble in 5% perchloric acid, are relatively small (molecular weight in the range of 10–16 kDa), and have both a high basic and a high acidic amino acid content. The amino acid constitution of these proteins is similar to that of the HMGB protein family of vertebrates. The proteins cross-react with antibodies raised against the HMGD chromosomal protein of Drosophila melanogaster. The possible relatedness of these proteins to high mobility group proteins is discussed.     

High mobility group (HMG) proteins in the tammar wallaby Macropus eugenii: quantitative variations between tissues and testis-specific co-extracted proteins

1. Tammar wallaby (Macropus eugenii, Marsupialia) proteins with similar electrophoretic mobilities to calf non-histone chromosomal proteins HMG 1, 2, 14 and 17 are perchloric acid extracted from whole tissues (liver, kidney, spleen, brain and testis) and purified liver nuclei (using PCA or 0.35 M NaCl). 2. Tammar and calf HMG 1 have similar amino acid compositions. 3. Two testis-specific basic proteins co-extracting with HMG-like proteins from both tammar and red kangaroo (Megaleia rufa) are found in whole testis, purified testis nuclei, but not epididymis. 4. Tammar HMG 2 separates into two components on both acid urea and SDS gels. The larger, more basic protein, HMG 2b, is relatively abundant in proliferating tissues (testis, spleen).     

Beyond linker histones and high mobility group proteins: global profiling of perchloric acid soluble proteins

Extraction with HClO(4) provides an easy method for efficient enrichment of both histone H1 and HMG proteins from a variety of tissues. Usually, the histone and the HMG proteins are the most abundant components of the extracts, however, other proteins have frequently been observed but only seldom studied in more detail. Here we describe a study aimed at global characterization of HClO(4) extractable proteins from breast cancer cell lines. We report identification of 150 unique proteins by liquid chromatography tandem mass spectrometry including almost all major histone H1 variants and canonical members of the HMG protein families. In the extracts, diverse proteins with HMG-like amino acid composition were identified and their post-translational modifications were mapped. Importantly, those include multiple proteins known or supposed to be related to cell proliferation and cancer. Since purification of these proteins as well as low abundant variants of histone and HMG proteins is difficult due to their metabolic instability, characterization of these proteins from crude extracts can facilitate studies aimed at better understanding of their function.     

HMG-like protein in barley and corn nuclei

Chromosomal proteins have been isolated from barley (Hordeum vulgare) and corn (Zea mays) nuclei by extraction with 5% perchloric acid. In each plant, one protein was shown to belong to the HMG proteins. Their molecular weights are very close to that of HMG 14 from chicken erythrocytes, as shown by electrophoretic mobility in SDS polyacrylamide gels. In acetic acid-urea-Triton polyacrylamide gels they migrate between HMG 1,2 and HMG 14, from chicken erythrocytes. Their amino acid compositions are typical of HMG proteins, with equivalent high values of acidic and basic residues. Extraction of HMG's from purified barley chromatin fractions with 0.35 M NaCl considerably reduces histone H2 contamination and increases the yield of HMG up to 0.7% of the total histones. In this technique a second protein was extracted which is soluble in 2% Trichloroacetic acid and shows electrophoretic mobility analogous to those of HMG 14 and 17 from chicken erythrocytes. Whether or not these proteins are counterparts of the animal HMG's 1–2 or HMG's 14–17 is discussed.     

Four differently chromatin‐associated maize HMG domain proteins modulate DNA structure and act as architectural elements in nucleoprotein complexes

In contrast to other eukaryotes which usually express two closely related HMG1-like proteins, plant cells have multiple relatively variable proteins of this type. A systematic analysis of the DNA-binding properties of four chromosomal HMG domain proteins from maize revealed that they bind linear DNA with similar affinity. HMGa, HMGc1/2 and HMGd specifically recognise diverse DNA structures such as DNA mini-circles and supercoiled DNA. They induce DNA-bending, and constrain negative superhelical turns in DNA. In the presence of DNA, the HMG domain proteins can self-associate, whereas they are monomeric in solution. The maize HMG1-like proteins have the ability to facilitate the formation of nucleoprotein structures to different extents, since they can efficiently replace a bacterial chromatin-associated protein required for the site-specific β-mediated recombination. A variable function of the HMG1-like proteins is indicated by their differential association with maize chromatin, as judged by their ‘extractability’ from chromatin with spermine and ethidium bromide. Collectively, these findings suggest that the various plant chromosomal HMG domain proteins could be adapted to act in different nucleoprotein structures in vivo.     

Insect proteins homologous to mammalian high mobility group protein 1. Characterization and DNA-binding properties.

Two chromosomal high mobility group (HMG) proteins from larvae of Chironomus thummi (Diptera) and from an epithelial cell line of Chironomus tentans were purified to homogeneity and chemically characterized. cDNA clones encoding these proteins were isolated from an expression library using an immunoscreening approach and were sequenced. The deduced amino acid sequences revealed their homology to HMG protein 1 of vertebrates. These insect proteins have therefore been designated cHMG1a and cHMG1b. They have a molecular mass of 12,915 and 12,019 kDa, respectively, and preferentially bind to AT-rich DNA. Indirect immunofluorescence microscopy with a polyclonal antibody showed the presence of cHMG1a and cHMG1b in condensed chromomeres but not in puffs, nucleoli, and cytoplasm. The cHMG1a and cHMG1b genes were both localized to a single band in region 14 of chromosome 1 of C. tentans and appear to be single copy genes. An immunologically related protein was purified from Drosophila melanogaster Kc cells. Its size and amino acid composition indicate that it is an HMG1 of D. melanogaster. On the other hand, our antibody did not recognize calf HMG1. The identification and characterization of HMG1 proteins in insects with polytene chromosomes opens new possibilities for studying function(s) of this group of chromosomal proteins.     

High-mobility group chromosomal proteins of wheat

Four proteins have been extracted from purified chromatin of wheat embryos with 0.35 M NaCl. These proteins are soluble in 2% (w/v) trichloroacetic acid and thus meet the original operational requirements to be classified as "high-mobility group" (HMG) chromosomal proteins. The proteins have been characterized by one- and two-dimensional electrophoresis, amino acid analysis, and peptide mapping. Three of the proteins (HMGb, c, and d) share the mammalian HMG characteristic of being rich in both acidic and basic amino acid residues. Unlike their putative mammalian counterparts, these plant HMG proteins contain less than 7 mol % proline. The fourth wheat protein (HMGa) is rich in both proline and in basic amino acid residues. This wheat protein, however, contains only about half the proportion of acidic residues found in mammalian HMG proteins--a characteristic also found in the trout testis HMG protein, H6. Comparative peptide maps show that none of the wheat HMG proteins are degradation products of other HMG proteins or the H1 histones. The peptide maps have not, however, been useful in establishing homologies with mammalian HMG proteins. Wheat HMG proteins are released from DNase I-treated nuclei and co-isolate with micrococcal nuclease-sensitive chromatin fractions. Similar observations concerning the HMG proteins of vertebrate animals have been considered consistent with a role for these proteins as structural components of actively transcribed chromatin.     

Extraction and partial characterization of non-histone nuclear proteins of Schistosoma mansoni.

A pool of nuclear proteins from adult worms of Schistosoma mansoni was analyzed for amino acid composition and found to be compatible with high mobility group (HMG) proteins. One of the schistosome HMG proteins was identified as HMG 2 by one-dimensional and two-dimensional PAGE. Stage-specific differences in the HMG-like protein composition were encountered when adult worms were compared to schistosomula, the larval form. Immobilization of the adult male and female nuclear proteins onto nitrocellulose, followed by hybridization against 32P-F-10, a schistosome sex specific gene encoding a major egg shell protein, revealed distinct banding patterns. On the other hand, a synthetic oligonucleotide, derived from the 3' untranslated end of the F-10 gene and possibly containing one regulatory element of the gene, bound mainly to male low MW proteins.     

DNA and histone H1 interact with different domains of HMG 1 and 2 proteins.

High mobility group (HMG) proteins 1 and 2 from calf thymus have been digested under structuring conditions (0.35 M NaCl, pH 7.1) with two proteases of different specificities, trypsin and V8. The two proteases give a different but restricted pattern of peptides in a time course digestion study. However, when the interactions of the peptides with DNA are studied by blotting, a closely related peptide from HMG-1 and -2 does not show any apparent binding. This peptide, from the V8 protease digestion, has been isolated by DNA-cellulose chromatography and has the amino acid composition predicted for a fragment containing the two C-terminal domains of the protein, i.e., approximately residues 74-243 for HMG-1. The same peptide shows the only interaction detectable with labelled histone H1. A separate function for the different domains of HMG proteins 1 and 2 is proposed.     

Sequence and characterization of 6 Lea proteins and their genes from cotton

Lea genes code for mRNAs and proteins that are late embryogenesis abundant in higher plant seed embryos. They appear to be ubiquitous in higher plants and may be induced to high levels of expression in other tissues and at other times of ontogeny by ABA and/or desiccation. Presented here are the genomic and cDNA sequences for 6 of these genes from cotton seed embryos and the derived amino acid sequences of the corresponding proteins.The Lea genes contain the standard sequence features of eucaryotic genes (TATA box and poly (A) addition sequences) and have 1 or more introns. Sequences differences between cDNA and genomic DNA confirm the existence of small multigene families for several Lea genes. The amino acid composition and sequence for the Lea proteins are unusual. Five are extremely hydrophilic, four contain no cys or trp and 4 have sequence domains that suggest amphiphilic helical structures. Hypothetical functions in desiccation survival, based on amino acid sequence, are discussed.     

High mobility group nonhistone chromosomal proteins of the developing sea urchin embryo

The high mobility group or HMG proteins are nonhistone chromosomal proteins that have been found in relatively high amounts in nuclei of many tissues. A number of studies have shown that some of these proteins are preferentially associated with actively transcribed regions of the genome and may play a role in maintaining these regions in an active state. In this study, we undertook an investigation of the high mobility group proteins from the sea urchin, Stronglyocentrotus purpuratus. Initially the putative sea urchin HMGs were extracted from isolated nuclei of hatching blastula-stage embryos with 5% perchloric acid (PCA). The major proteins in this extract were characterized according to their electrophoretic mobility, amino acid composition, and association with isolated deoxyribonucleoprotein particles. The results indicate there is only one "major" sea urchin HMG protein, termed P2 in this paper. An estimate of the amount of P2 in relation to the inner histones, however, was low compared to what has been found for other HMG proteins. Of the other major 5% PCA-extractable proteins, one was identified as the cleavage stage H1. Another protein apparently resulted from H3 contamination in the 5% PCA extract, and the fourth major protein did not have all the characteristics of an HMG. In particular, it was not found associated with nucleosomal particles. The HMG proteins from other developmental stages were then examined. Five percent PCA extracts of nuclei from unfertilized eggs, 2-cell, 16-cell, hatching blastula, gastrula, and pluteus stages were analyzed on SDS- and acetic acid-urea gels. This analysis indicated that P2 exists in two different forms differing slightly in charge. The less basic form was found in the egg, 2-cell and 16-cell extracts. At the hatching blastula stage, both forms were present and by pluteus stage, the more basic form predominated. It appears that P2 is undergoing a developmental change from a less to more basic form. The presence of P2 in the 5% PCA extract of egg nuclei is proof that P2 does not initially appear sometime during embryogenesis but is already in the egg nucleus prior to fertilization.     

Tetrahymena contain two distinct and unusual high mobility group (HMG)- like proteins

Previous studies have described the existence of high mobility group (HMG)-like proteins in macronuclei of the ciliated protozoan, Tetrahymena thermophila (Hamana, K., and K. Iwai, 1979, J. Biochem. [Tokyo], 69:1097-1111; Levy-Wilson, B., M. S. Denker, and E. Ito, 1983, Biochemistry, 22:1715-1721). In this report, two of these proteins, LG- 1 and LG-2, have been further characterized. Polyclonal antibodies raised against LG-1 and LG-2 fail to cross react with each other or any other macronuclear polypeptide in immunoblotting analyses. As well, LG- 1 and LG-2 antibodies do not react with calf thymus, chicken, or yeast HMG proteins. Consistent with these results, a 47 amino-terminal sequence of LG-1 has been determined that shows limited homology to both calf thymus HMGs 1 and 2 and HMGs 14 and 17. Two internal sequences of V8 protease-generated peptides from LG-2 have been determined, and these do not share any homology to the LG-1 sequence or any other sequenced HMG proteins. Comparison of the partial sequences of LG-1 and LG-2 with the complete amino acid sequence of the Tetrahymena histone H1 (Wu, M., C. D. Allis, R. Richman, R. G. Cook, and M. A. Gorovsky, 1986, Proc. Natl. Acad. Sci. USA, 83:8674-8678) rules out the possibility that LG-1 and LG-2 are proteolytically derived from H1, the other major macronuclear perchloric acid-soluble protein. Interestingly, however, both LG-1 and LG-2 are efficiently extracted from macronuclei by elutive intercalation (Schroter, H., G. Maier, H. Ponsting, and A. Nordheim, 1985, Embo (Eur. Mol. Biol. Organ.) J., 4:3867-3872), suggesting that both may share yet undetermined properties with HMGs 14 and 17 of higher eukaryotes. Examination of the pattern of LG-1 and LG-2 synthesis during the sexual phase of the life cycle, conjugation, demonstrates that the synthesis of LG-1 and LG-2 is coordinately increased from basal levels during the differentiation of new macronuclei (7-13 h), suggesting that both of these proteins play a role in determining a macronuclear phenotype. However, a specific induction of LG-2 synthesis is detected in early stages of conjugation (meiotic prophase, 1-4 h), leading to maximal synthesis of LG-2 at 3 h. Interestingly, the early induction of LG-2 synthesis closely parallels the hyperphosphorylation of histone H1. Taken together, these data suggest that LG-1 and LG-2 are not strongly related to each other or to higher eukaryotic HMG proteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

DNA binding by single HMG box model proteins

The HMG1/2 family is a large group of proteins that share a conserved sequence of ~80 amino acids rich in basic, aromatic and proline side chains, referred to as an HMG box. Previous studies show that HMG boxes can bind to DNA in a structure-specific manner. To define the basis for DNA recognition by HMG boxes, we characterize the interaction of two model HMG boxes, one a structure-specific box, rHMGb from the rat HMG1 protein, the other a sequence-specific box, Rox1 from yeast, with oligodeoxynucleotide substrates. Both proteins interact with single-stranded oligonucleotides in this study to form 1:1 complexes. The stoichiometry of binding of rHMGb to duplex or branched DNAs differs: for a 16mer duplex we find a weak 2:1 complex, while a 4:1 protein:DNA complex is detected with a four-way DNA junction of 16mers in the presence of Mg²⁺. In the case of the sequence-specific Rox1 protein we find tight 1:1 and 2:1 complexes with its cognate duplex sequence and again a 4:1 complex with four-way branched DNA. If the DNA branching is reduced to three arms, both proteins form 3:1 complexes. We believe that these multimeric complexes are relevant for HMG1/2 proteins in vivo, since Mg²⁺ is present in the nucleus and these proteins are expressed at a very high level.