非洲爪蟾孵化酶的cDNA结构及其部分生物化学特性的研究

以UVS．2为探针从第25期非洲爪蟾胚胎头背部的cDNA文库中筛选出了一个1．8kb的孵化酶基因(xhe),其转录产物最早出现于第17期胚胎的头背部,在第30期转录量达到高峰,随后便逐渐减少。该基因含有编码514个氨基酸的一个开框阅读框架,含有信号肽和原酶序列。所推测出的成熟蛋白有425个氨基酸,包括位于N一端的含有200个氨基酸的金属蛋白酶序列和位于C端的两个各110个氨基酸的CUB重复区。而UVS．2只代表该基因C端大约3／4的部分。同时还发现该酶分子量为60kDa,是一种胰蛋白酶类型的金属蛋白酶。它很不稳定,在纯化过程中极易降解为40kDa分子。60kDa分子具有很强的卵黄膜溶解活性和蛋白酶活性。其中CUB重复区很可能在介导卵黄膜和40kDa分子中起着重要作用,而40kDa分子很可能是在纯化操作过程中,由60kDa分子发生降解或自身降解丢失了两个CUB重复区而形成的,它只是60kDa分子中的一个金属蛋白酶主功能区,所以它没有卵黄膜溶解活性,尽管仍具有很强的蛋白酶活性。     

Conserved DNA binding and self-association domains of the Drosophila zeste protein.

The zeste gene product is involved in two types of genetic effects dependent on chromosome pairing: transvection and the zeste-white interaction. Comparison of the predicted amino acid sequence with that of the Drosophila virilis gene shows that several blocks of amino acid sequence have been very highly conserved. One of these regions corresponds to the DNA binding domain. Site-directed mutations in this region indicate that a sequence resembling that of the homeodomain DNA recognition helix is essential for DNA binding activity. The integrity of an amphipathic helical region is also essential for binding activity and is likely to be responsible for dimerization of the DNA binding domain. Another very strongly conserved domain of zeste is the C-terminal region, predicted to form a long helical structure with two sets of heptad repeats that constitute two long hydrophobic ridges at opposite ends and on opposite faces of the helix. We show that this domain is responsible for the extensive aggregation properties of zeste that are required for its role in transvection phenomena. A model is proposed according to which the hydrophobic ridges induce the formation of open-ended coiled-coil structures holding together many hundreds of zeste molecules and possibly anchoring these complexes to other nuclear structures.     

Multiple functional domains of Tat, the trans-activator of HIV-1, defined by mutational analysis.

The tat gene of HIV-1 is a potent trans-activator of gene expression from the HIV long terminal repeat (LTR). To define the functionally important regions of the product of the tat gene (Tat) of HIV-1, deletion, linker insertion and single amino acid substitution mutants within the Tat coding region of strain SF2 were constructed. The effect of these mutations on trans-activation was assessed by measuring the expression of the bacterial chloramphenicol acetyltransferase (CAT) reporter gene linked to the HIV-LTR. These studies have revealed that four different domains of the protein that map within the N-terminal 56 amino acid region are essential for Tat function. In addition to the essential domains, an auxiliary domain that enhances the activity of the essential region has also been mapped between amino acid residues 58 and 66. One of the essential domains maps in the N-terminal 20 amino acid region. The other three essential domains are highly conserved among the various strains of HIV-1 and HIV-2 as well as simian immunodeficiency virus (SIV). Of the conserved domains, one contains seven Cys residues and single amino acid substitutions for several Cys residues indicate that they are essential for Tat function. The second conserved domain contains a Lys X Leu Gly Ile X Tyr motif in which the Lys residue is essential for trans-activation and the other residues are partially essential. The third conserved domain is strongly basic and appears to play a dual role. Mutants lacking this domain are deficient in trans-activation and in efficient targeting of Tat to the nucleus and nucleolus. The combination of the four essential domains and the auxiliary domain contribute to the near full activity observed with the 101 amino acid Tat protein.     

A tandemly repeated sequence determines the binding domain for an erythrocyte receptor binding protein of P. falciparum

Erythrocyte invasion by the malarial merozoite is a receptor-mediated process, an obligatory step in the development of the parasite. The Plasmodium falciparum protein GBP-130, which binds to the erythrocyte receptor glycophorin, is shown here to encode the binding site in a domain composed of a tandemly repeated 50 amino acid sequence. The amino acid sequence of GBP-130, deduced from the cloned and sequenced gene, reveals that the protein contains 11 highly conserved 50 amino acid repeats and a charged N-terminal region of 225 amino acids. Binding studies on recombinant proteins expressing different numbers of repeats suggest that a correlation exists between glycophorin binding and repeat number. Thus, a repeat domain, a common feature of plasmodial antigens, has been shown to have a function independent of the immune system. This conclusion is further supported by the ability of antibodies directed against the repeat sequence to inhibit the in vitro invasion of erythrocytes by merozoites.     

Cloning and expression of cDNA encoding a neural calcium-dependent cell adhesion molecule: its identity in the cadherin gene family

The neural cadherin (N-cadherin) is a Ca2+-dependent cell-cell adhesion molecule detected in neural tissues as well as in non-neural tissues. We report here the nucleotide sequence of the chicken N-cadherin cDNA and the deduced amino acid sequence. The sequence data suggest that N-cadherin has one transmembrane domain which divides the molecule into an extracellular and a cytoplasmic domain; the extracellular domain contains internal repeats of characteristic sequences. When the N-cadherin cDNA connected with virus promoters was transfected into L cells which have no endogenous N-cadherin, the transformants acquired the N-cadherin-mediated aggregating property, indicating that the cloned cDNA contained all information necessary for the cell-cell binding action of this molecule. We then compared the primary structure of N-cadherin with that of other molecules defined as cadherin subclasses. The results showed that these molecules contain common amino acid sequences throughout their entire length, which confirms our hypothesis that cadherins make a gene family.     

Studies on the structure-function relationship of the HNK-1 associated glucuronyltransferase, GlcAT-P, by computer modeling and site-directed mutagenesis

All members of a glucuronyltransferase (GlcAT) gene family cloned to date contain four conserved regions (modules I-IV), which are widely located in the catalytic domain. In order to understand the biological significance of these modules, we investigated the structure-function relationship of GlcAT-P by means of the combination of site-directed mutagenesis and computer aided three-dimensional modeling. The wild-type and mutant GlcAT-Ps were expressed in Escherichia coli as glutathione-S-transferase (GST)-fused soluble proteins. Most of the mutants in which a polar amino acid within the modules was replaced with alanine lost their transferase activity almost completely, while all of the mutants in which the replacement was outside these modules retained the original catalytic activity. A three-dimensional (3-D) model of GlcAT-P was constructed by computer simulation with the three-dimensional structure of adenylate kinase (1AKE) as a template. This model predicted that the large catalytic domain of GlcAT-P forms a globular shape with a Rossmann-fold motif consisting of five alpha-helix and beta-sheet repeats. The putative catalytic pocket consisting mainly of modules I-III is surrounded by a cluster of polar amino acids, which are essential for the transferase activity and also for the binding to the acceptor substrate (essential amino acids), asialo-orosomucoid. There is the second cluster of essential amino acids almost on the opposite surface of the molecule, in which an aspartic acid repeat (DDD) is located. The biological significance of the second cluster is currently not clear but it may be associated with the interaction of the enzyme with modulation molecules, manganese and membrane phospholipids.     

Primary structure of the human heparan sulfate proteoglycan from basement membrane (HSPG2/perlecan). A chimeric molecule with multiple domains homologous to the low density lipoprotein receptor, laminin, neural cell adhesion molecules, and epidermal growth factor.

We have determined the complete nucleotide and deduced amino acid sequence of the major protein core of the human heparan sulfate proteoglycan HSPG2/perlecan of basement membranes. Eighteen overlapping cDNA clones comprise 14.35 kilobase pairs (kb) of contiguous sequence with an open reading frame of 13.2 kb. The mature protein core, without the signal peptide of 21 amino acids, has a M(r) of 466,564. This large protein is composed of multiple modules homologous to the receptor of low density lipoprotein, laminin, neural cell adhesion molecules, and epidermal growth factor. Domain I, near the amino terminus, appears unique for the proteoglycan since it shares no significant homology with any other proteins. It contains three Ser-Gly-Asp sequences that could act as attachment sites for heparan sulfate glycosaminoglycans. Domain II is highly homologous to the LDL receptor and contains four repeats with perfect conservation of all 6 consecutive cysteines. Next is domain III which shares homology to the short arm of laminin A chain and contains four cysteine-rich regions intercalated among three globular domains. Domain IV, the largest module with greater than 2000 residues, contains 21 repeats of the immunoglobulin type as found in neural cell adhesion molecule. Near the beginning of this domain, there is a stretch of 29 hydrophobic amino acids which could allow the molecule to interact with the plasma membrane. Domain V, similar to the carboxyl-terminal globular G-domain of laminin A and to the related protein merosin, contains three globular regions and four EGF-like repeats. In situ hybridization and immunoenzymatic studies show a close association of this gene product with a variety of cells involved in the assembly of basement membranes, in addition to being localized within the stromal elements of various connective tissues. Our studies show that this proteoglycan is present in all vascularized tissues and suggest that this unique molecule has evolved from the utilization of modular structures with adhesive and growth regulatory properties.     

Human basement membrane heparan sulfate proteoglycan core protein: a 467-kD protein containing multiple domains resembling elements of the low density lipoprotein receptor, laminin, neural cell adhesion molecules, and epidermal growth factor

The primary structure of the large human basement membrane heparan sulfate proteoglycan (HSPG) core protein was determined from cDNA clones. The cDNA sequence codes for a 467-kD protein with a 21-residue signal peptide. Analysis of the amino acid sequence showed that the protein consists of five domains. The amino-terminal domain I contains three putative heparan sulfate attachment sites; domain II has four LDL receptor-like repeats; domain III contains repeats similar to those in the short arms of laminin; domain IV has lg-like repeats resembling those in neural cell adhesion molecules; and domain V contains sequences resembling repeats in the G domain of the laminin A chain and repeats in the EGF. The domain structure of the human basement membrane HSPG core protein suggests that this mosaic protein has evolved through shuffling of at least four different functional elements previously identified in other proteins and through duplication of these elements to form the functional domains. Comparison of the human amino acid sequence with a partial amino acid sequence from the corresponding mouse protein (Noonan, D. M., E. A. Horigan, S. R. Ledbetter, G. Vogeli, M. Sasaki, Y. Yamada, and J. R. Hassell. 1988. J. Biol. Chem. 263:16379-16387) shows a major difference between the species in domain IV, which contains the Ig repeats: seven additional repeats are found in the human protein inserted in the middle of the second repeat in the mouse sequence. This suggests either alternative splicing or a very recent duplication event in evolution. The multidomain structure of the basement membrane HSPG implies a versatile role for this protein. The heparan sulfate chains presumably participate in the selective permeability of basement membranes and, additionally, the core protein may be involved in a number of biological functions such as cell binding, LDL-metabolism, basement membrane assembly, calcium binding, and growth- and neurite-promoting activities.     

Mutational analysis of bovine papillomavirus type 1 E5 peptide domains involved in induction of cellular DNA synthesis.

Early gene E5 of bovine papillomavirus type 1 encodes a 44-amino-acid protein whose expression can transform immortalized mouse cell lines. We have previously reported that a chemically synthesized E5 peptide functions to induce cellular DNA synthesis upon microinjection into growth-arrested mouse cells. We further defined the two E5 domains essential for the full DNA synthesis induction activity by the analysis of E5 deletion and amino acid substitution mutant peptides. The first domain is the C-terminal 13-amino-acid core which is sufficient to activate DNA synthesis at high peptide concentration and contains two essential, highly conserved cysteine residues. The second domain is the 7-amino-acid hydrophobic sequence contiguous to the core domain which is sufficient to confer a 1,000-fold higher molar specific activity to the E5 peptide. A random hydrophobic sequence, but not charged amino acids, fulfills the function of the second domain.     

Internal symmetry of the mirror type and repeats in molecules of the membrane-bound forms of adenylate cyclase]

The analysis of mirror type internal symmetry distribution in primary structures of different types of mammalian membrane-bound adenylyl cyclases was made. The transmembrane domain clusters determining enzyme topology in membrane, a highly conservative region of cytoplasmic domains forming both catalytic and regulatory centres of adenylyl cyclases, and the functionally important regions in variable parts of their molecules (in particular, calmodulin binding regions) are shown to have symmetrical structures. These data are in conformity with a hypothesis put forward by the authors: the centres of internal symmetry may commonly either coincide with sites responsible for protein biological activity, or be spaced in the immediate vicinity of these sites. In different types of adenylyl cyclases long repeating sequences were identified. The segmentary structures were established for some enzyme subdomains. The regions containing repeats usually displayed a symmetrical structure which confirms a positive correlation between internal symmetry of amino acid sequence and its repeat distribution.     

Engineering ATPase activity in the isolated ABC cassette of human TAP1

The human transporter associated with antigen processing (TAP) translocates antigenic peptides from the cytosol into the endoplasmic reticulum lumen. The functional unit of TAP is a heterodimer composed of the TAP1 and TAP2 subunits, both of which are members of the ABC-transporter family. ABC-transporters are ATP-dependent pumps, channels, or receptors that are composed of four modules: two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). Although the TMDs are rather divergent in sequence, the NBDs are conserved with respect to structure and function. Interestingly, the NBD of TAP1 contains mutations at amino acid positions that have been proposed to be essential for catalytic activity. Instead of a glutamate, proposed to act as a general base, TAP1 contains an aspartate and a glutamine instead of the conserved histidine, which has been suggested to act as the linchpin. We used this degeneration to evaluate the individual contribution of these two amino acids to the ATPase activity of the engineered TAP1-NBD mutants. Based on our results a catalytic hierarchy of these two fundamental amino acids in ATP hydrolysis of the mutated TAP1 motor domain was deduced.     

PSI+] maintenance is dependent on the composition, not primary sequence, of the oligopeptide repeat domain

[PSI(+)], the prion form of the yeast Sup35 protein, results from the structural conversion of Sup35 from a soluble form into an infectious amyloid form. The infectivity of prions is thought to result from chaperone-dependent fiber cleavage that breaks large prion fibers into smaller, inheritable propagons. Like the mammalian prion protein PrP, Sup35 contains an oligopeptide repeat domain. Deletion analysis indicates that the oligopeptide repeat domain is critical for [PSI(+)] propagation, while a distinct region of the prion domain is responsible for prion nucleation. The PrP oligopeptide repeat domain can substitute for the Sup35 oligopeptide repeat domain in supporting [PSI(+)] propagation, suggesting a common role for repeats in supporting prion maintenance. However, randomizing the order of the amino acids in the Sup35 prion domain does not block prion formation or propagation, suggesting that amino acid composition is the primary determinant of Sup35's prion propensity. Thus, it is unclear what role the oligopeptide repeats play in [PSI(+)] propagation: the repeats could simply act as a non-specific spacer separating the prion nucleation domain from the rest of the protein; the repeats could contain specific compositional elements that promote prion propagation; or the repeats, while not essential for prion propagation, might explain some unique features of [PSI(+)]. Here, we test these three hypotheses and show that the ability of the Sup35 and PrP repeats to support [PSI(+)] propagation stems from their amino acid composition, not their primary sequences. Furthermore, we demonstrate that compositional requirements for the repeat domain are distinct from those of the nucleation domain, indicating that prion nucleation and propagation are driven by distinct compositional features.     

AcmA of Lactococcus lactis is an N-acetylglucosaminidase with an optimal number of LysM domains for proper functioning

AcmA, the major autolysin of Lactococcus lactis MG1363 is a modular protein consisting of an N-terminal active site domain and a C-terminal peptidoglycan-binding domain. The active site domain is homologous to that of muramidase-2 of Enterococcus hirae, however, RP-HPLC analysis of muropeptides released from Bacillus subtilis peptidoglycan, after digestion with AcmA, shows that AcmA is an N-acetylglucosaminidase. In the C-terminus of AcmA three highly similar repeated regions of 45 amino acid residues are present, which are separated by short nonhomologous sequences. The repeats of AcmA, which belong to the lysine motif (LysM) domain family, were consecutively deleted, removed, or, alternatively, one additional repeat was added, without destroying the cell wall-hydrolyzing activity of the enzyme in vitro, although AcmA activity was reduced in all cases. In vivo, proteins containing no or only one repeat did not give rise to autolysis of lactococcal cells, whereas separation of the producer cells from the chains was incomplete. Exogenously added AcmA deletion derivatives carrying two repeats or four repeats bound to lactococcal cells, whereas the derivative with no or one repeat did not. In conclusion, these results show that AcmA needs three LysM domains for optimal peptidoglycan binding and biological functioning.     

The globular domains of type VI collagen are related to the collagen-binding domains of cartilage matrix protein and von Willebrand factor.

Type VI collagen is a transformation-sensitive glycoprotein of the extracellular matrix of fibroblasts. We have isolated and sequenced several overlapping cDNA clones (4153 bp) which encode the entire alpha 2 subunit of chicken type VI collagen. The deduced amino acid sequence predicts that the alpha 2(VI) polypeptide consists of 1015 amino acid residues that are arranged in four domains: a hydrophobic signal peptide of 20 residues, an amino-terminal globular domain of 228 residues, a collagenous segment of 335 residues and a carboxy-terminal globular domain of 432 residues. The collagenous domain contains seven Arg-Gly-Asp tripeptide units, some of which are likely to be used as cell-binding sites. The globular domains contain three homologous repeats with an average length of 180 amino acid residues. These repeats show a striking similarity to the collagen-binding motifs found in von Willebrand factor and cartilage matrix protein. We therefore speculate that the globular domains of the alpha 2(VI) polypeptide may interact with collagenous structures.     

Novel dendritic kinesin sorting identified by different process targeting of two related kinesins: KIF21A and KIF21B

Neurons use kinesin and dynein microtubule-dependent motor proteins to transport essential cellular components along axonal and dendritic microtubules. In a search for new kinesin-like proteins, we identified two neuronally enriched mouse kinesins that provide insight into a unique intracellular kinesin targeting mechanism in neurons. KIF21A and KIF21B share colinear amino acid similarity to each other, but not to any previously identified kinesins outside of the motor domain. Each protein also contains a domain of seven WD-40 repeats, which may be involved in binding to cargoes. Despite the amino acid sequence similarity between KIF21A and KIF21B, these proteins localize differently to dendrites and axons. KIF21A protein is localized throughout neurons, while KIF21B protein is highly enriched in dendrites. The plus end-directed motor activity of KIF21B and its enrichment in dendrites indicate that models suggesting that minus end-directed motor activity is sufficient for dendrite specific motor localization are inadequate. We suggest that a novel kinesin sorting mechanism is used by neurons to localize KIF21B protein to dendrites since its mRNA is restricted to the cell body.     

MosA, a protein implicated in rhizopine biosynthesis in Sinorhizobium meliloti L5-30, is a dihydrodipicolinate synthase

MosA is a gene product encoded on a pSym megaplasmid of Sinorhizobium meliloti L5-30. The gene is part of an operon reported to be essential for the synthesis of the rhizopine 3-O-methyl-scyllo-inosamine. MosA has been assigned the function of an O-methyltransferase. However, the reported sequence of this protein is very much like that of dihydrodipicolinate synthase (DHDPS), except for a 40 amino acid residue C-terminal domain. This similarity contradicts accepted ideas regarding structure-function relationships of enzymes. We have cloned and overexpressed the recombinant gene in Escherichia coli, and discovered that the reported sequence contains an error resulting in a frame-shift. The correct sequence contains a new stop codon, truncating the C-terminal 41 amino acid residues of the reported sequence. The expressed protein, bearing an N-terminal polyhistidine tag, catalyzes the condensation of pyruvate and aspartate beta-semialdehyde efficiently, suggesting that this activity is not a side-reaction, but an activity for which this enzyme has evolved. Electro-spray mass spectrometry experiments and inhibition by L-lysine are consistent with the enzyme being a DHDPS. E.coli AT997, a mutant host normally requiring exogenous diaminopimelate for growth, could be complemented by transformation with a plasmid bearing the gene encoding MosA. A role for this enzyme in rhizopine synthesis cannot be ruled out, but is called into question.     

Investigating the structure of human RNase H1 by site-directed mutagenesis

In this study we examine for the first time the roles of the various domains of human RNase H1 by site-directed mutagenesis. The carboxyl terminus of human RNase H1 is highly conserved with Escherichia coli RNase H1 and contains the amino acid residues of the putative catalytic site and basic substrate-binding domain of the E. coli RNase enzyme. The amino terminus of human RNase H1 contains a structure consistent with a double-strand RNA (dsRNA) binding motif that is separated from the conserved E. coli RNase H1 region by a 62-amino acid sequence. These studies showed that although the conserved amino acid residues of the putative catalytic site and basic substrate-binding domain are required for RNase H activity, deletion of either the catalytic site or the basic substrate-binding domain did not ablate binding to the heteroduplex substrate. Deletion of the region between the dsRNA-binding domain and the conserved E. coli RNase H1 domain resulted in a significant loss in the RNase H activity. Furthermore, the binding affinity of this deletion mutant for the heteroduplex substrate was approximately 2-fold tighter than the wild-type enzyme suggesting that this central 62-amino acid region does not contribute to the binding affinity of the enzyme for the substrate. The dsRNA-binding domain was not required for RNase H activity, as the dsRNA-deletion mutants exhibited catalytic rates approximately 2-fold faster than the rate observed for wild-type enzyme. Comparison of the dissociation constant of human RNase H1 and the dsRNA-deletion mutant for the heteroduplex substrate indicates that the deletion of this region resulted in a 5-fold loss in binding affinity. Finally, comparison of the cleavage patterns exhibited by the mutant proteins with the cleavage pattern for the wild-type enzyme indicates that the dsRNA-binding domain is responsible for the observed strong positional preference for cleavage exhibited by human RNase H1.     

Mutagenesis studies on the amino acid residues involved in the iron-binding and the activity of human 5-lipoxygenase.

Human 5-lipoxygenase contains a non-heme iron essential for its activity. In order to determine which amino acid residues are involved in the iron-binding and the lipoxygenase activity, nine amino acid residues in highly homologous regions among the lipoxygenases were individually replaced by means of site-directed mutagenesis. Mutant 5-lipoxygenases in which His-367 or His-550 was replaced by either Asn or Ala, His-372 by either Asn or Ser, or Glu-376 by Gln were completely devoid of the activity. Though mutants containing an alanine residue instead of His-390 or His-399 lacked the activity, the corresponding asparagine substituted mutants exhibited. The other mutants retained the enzyme activity. These results strongly suggest that His-367, His-372, His-550 and Glu-376 are crucial for 5-lipoxygenase activity and coordinate to the essential iron.