Genomic organization of the serine protease light chain of mouse complement factor I gene

A portion of the mouse complement factor I (mCFI) gene encoding for the mCFI light chain was cloned from a mouse 129/SVJ1 bacterial artificial chromosome library. It contains five exons and four introns. The intron sizes are remarkably different from the human homolog. Several polymorphisms were found in exon 13. One polymorphism was in the coding region, which causes a threonine in the Balb/c mCFI to be replaced by an isoleucine in the 129/SVJ1 mCFI. The other two polymorphisms are located in the 3' untranslated region. The organization of the serine protease domain in mCFI is similar to that of trypsin but very different from that of the other complement serine proteases.     

Different evolutionary histories of kringle and protease domains in serine proteases: A typical example of domain evolution

With the aim of elucidating the evolutionary processes of the kringle and protease domains in serine proteases which are involved with the system of blood coagulation and fibrinolysis, we constructed phylogenetic trees for the kringle and protease domains, separately, by use of amino acid sequence data. The phylogenetic trees constructed clearly showed that the topologies were different between the kringle and protease domains. Because both domains are coded by single peptides of serine proteases, this strongly suggests that the kringle and protease domains must have undergone different evolutionary processes. Thus, these observations imply that serine proteases evolve in a way such that each domain is a unit of evolution, exemplifying a typical mode of domain evolution. A possible relationship between the domain evolution and the exon shuffling theory is also discussed from the viewpoint of gene evolution.     

Structure-function analysis of hepatocyte growth factor: identification of variants that lack mitogenic activity yet retain high affinity receptor binding.

Hepatocyte growth factor (HGF) is a potent mitogen for parenchymal liver, epithelial and endothelial cells. Structurally, it has similarities to kringle-containing serine proteases, although it does not possess proteolytic activity. A structure-activity relationship study of human HGF was performed by functional analysis of HGF substitution and deletion variants. Analysis of HGF variants was accomplished by defining their ability to induce DNA synthesis on hepatocytes in primary culture and to compete with wild-type HGF for binding to a soluble form of the HGF receptor. Three groups of variants were made: (i) substitutions at the cleavage site, (ii) substitutions within the protease-like domain and (iii) deletions of the beta-chain and/or kringle domains. Our results show that: (i) single-chain HGF is a zymogen-like promitogen in that cleavage into a two-chain form is required for biological activity, however, the single chain form of HGF still retains substantial receptor binding capacity; (ii) certain mutations in the protease-like domain result in variants that are completely defective for mitogenic activity, yet exhibit apparent receptor binding affinities similar to wild-type HGF (Kd approximately 50-70 pM); and (iii) a variant containing the N-terminal 272 residues of mature HGF showed only a 4-fold increase in Kd when compared with wild-type HGF indicating that a primary receptor binding determinant is located within this sequence.     

Characterization of the DNF15S2 locus on human chromosome 3: identification of a gene coding for four kringle domains with homology to hepatocyte growth factor.

A human genomic DNA library was screened by using conditions of reduced stringency with a bovine cDNA probe coding for the kringle domains in prothrombin in order to isolate the human prothrombin gene. Twelve positives were identified, three of which coded for prothrombin (Degen & Davie, 1987). Phage L5 was characterized in more detail because of its strong hybridization to the cDNA probe and its unique restriction map compared to the gene coding for human prothrombin. The gene in L5 was sequenced and found to code for a kringle-containing protein. A human liver cDNA library was screened by using a genomic probe from the gene in L5. cDNAs were isolated that contained sequence identical with regions in the gene in L5. Comparison of the cDNA with the gene indicated that the gene in L5 was composed of 18 exons separated by 17 intervening sequences and is 4690 bp in length. Exons ranged in size from 36 to 242 bp in length while intervening sequences ranged from 77 to 697 bp in length. The putative protein encoded by the gene in L5 contains four kringle domains followed by a serine protease-like domain. This domain structure is identical with that found in hepatocyte growth factor (HGF), although the two proteins are only about 50% identical. On the basis of the similarity of the protein encoded by L5 and HGF, we propose that the putative L5 protein be tentatively called HGF-like protein until a function is identified. The DNA sequence of the gene and cDNA and its translated amino acid sequence were compared against GenBank and NBRF databases. Sequences homologous to DNF15S1 and DNF15S2, human DNF15S2 lung mRNA, and rat acyl-peptide hydrolase were identified in exon 17 to the 3' end of the characterized sequence for the gene. From our results, it is apparent that the gene coding for human HGF-like protein is located at the DNF15S2 locus on human chromosome 3 (3p21). The gene for acyl-peptide hydrolase is 444 bp downstream of the gene coding for HGF-like protein, but on the complementary strand. The DNF15S2 locus has been proposed to code for one or more tumor suppressor genes since this locus is deleted in DNA from small cell lung carcinoma, other lung cancers, renal cell carcinoma, and von Hippel-Lindau syndrome.     

Structure of the human neutrophil elastase gene

The gene for human neutrophil elastase (NE), a powerful serine protease carried by blood neutrophils and capable of destroying most connective tissue proteins, was cloned from a genomic DNA library of a normal individual. The NE gene consists of 5 exons and 4 introns included in a single copy 4-kilobase segment of chromosome 11 at q14. The coding exons of the NE gene predict a primary translation product of 267 residues including a 29-residue N-terminal precursor peptide and a 20-residue C-terminal precursor peptide. Analysis of the N-terminal peptide sequence suggests it contains a 27-residue "pre" signal peptide followed by a "proN" dipeptide, similar to that of other blood cell lysosomal proteases. The sequences for the mature 218-residue NE protein are included in exons II-V. The 5'-flanking region of the gene includes typical TATA, CAAT, and GC sequences within 61 base pairs (bp) of the cap site. The sequence 1.5 kilobases 5' to exon I contains several interesting repetitive sequences including six tandem repeats of unique 52- or 53-bp sequences. The 5'-flanking region also contains a 19-bp segment with 90% homology to a segment of the 5'-flanking region of the human myeloperoxidase (MPO) gene, a gene also expressed in bone marrow precursor cells and a protein stored in the same neutrophil granules as NE. In addition, like the MPO gene, the NE 5'-flanking region has several regions with greater than or equal to 75% homology to sequences 5' to c-myc, but there is no overlap between the NE-c-myc and MPO-c-myc homologous sequences.     

Crystal structure of the HGF beta-chain in complex with the Sema domain of the Met receptor

The Met tyrosine kinase receptor and its ligand, hepatocyte growth factor (HGF), play important roles in normal development and in tumor growth and metastasis. HGF-dependent signaling requires proteolysis from an inactive single-chain precursor into an active alpha/beta-heterodimer. We show that the serine protease-like HGF beta-chain alone binds Met, and report its crystal structure in complex with the Sema and PSI domain of the Met receptor. The Met Sema domain folds into a seven-bladed beta-propeller, where the bottom face of blades 2 and 3 binds to the HGF beta-chain 'active site region'. Mutation of HGF residues in the area that constitutes the active site region in related serine proteases significantly impairs HGF beta binding to Met. Key binding loops in this interface undergo conformational rearrangements upon maturation and explain the necessity of proteolytic cleavage for proper HGF signaling. A crystallographic dimer interface between two HGF beta-chains brings two HGF beta:Met complexes together, suggesting a possible mechanism of Met receptor dimerization and activation by HGF.     

Complement genes C1r and C1s feature an intronless serine protease domain closely related to haptoglobin

The exon-intron structure of the human complement C1s gene displays a striking similarity with that of the gene encoding haptoglobin, a peculiar transport protein distantly related to the serine proteases. While the protease regions of the serine zymogens are typically encoded by multiple exons, the protease domains of C1s and of its genetically linked and functionally interacting homolog C1r are encoded as intronless domains, not unlike a region of haptoglobin, which in fact is devoid of proteolytic activity. The close similarity of the C1s gene with haptoglobin includes the precise conservation of exon-intron junctions and it extends to upstream exons encoding the short repeats typical of several complement components, but found also in other functionally unrelated proteins. Additional evidence of the common ancestry of C1r, C1s and haptoglobin is the presence, within the protease domain, of a set of sequence markers that distinguish these three proteins from all known serine proteases. The finding of vertebrate serine protease genes with an uninterrupted protease-encoding exon supports the definition of a novel evolutionary branch of this gene family and rules out the hypothesis that regards this unusual exon as an irrelevant byproduct of the extravagant functional divergence of haptoglobin.     

Partial nucleotide sequence of a novel bovine major histocompatibility complex class II β-chain gene, BoLA-DIB

A bovine genomic clone that hybridized to HLA-DQ beta cDNA was isolated and fragments containing the beta 1, beta 2 and transmembrane (TM) exons subcloned. The nucleotide sequences of the exons and flanking intron regions were determined. Comparisons of these exon nucleotide sequences and derived amino acid sequences to human class II beta-chain sequences showed that this gene is only 77% identical to HLA-DQ beta and about 75% identical to bovine DQ beta-like genes. The exon sequences were more divergent from other class II beta-chain genes. However, structural features such as conserved cysteines and regions of amino acids strongly suggest this to be a class II beta-chain gene. When exon-containing fragments were used as hybridization probes on Southern blots of bovine genomic DNA digested with Eco RI or Pvu II, each exon hybridized to a single band. Based on these results we have referred to this gene as a novel bovine class II beta-chain gene, BoLA-DIB.     

Structural and functional basis of the serine protease-like hepatocyte growth factor beta-chain in Met binding and signaling

Hepatocyte growth factor (HGF), a plasminogen-related growth factor, is the ligand for Met, a receptor tyrosine kinase implicated in development, tissue regeneration, and invasive tumor growth. HGF acquires signaling activity only upon proteolytic cleavage of single-chain HGF into its alpha/beta heterodimer, similar to zymogen activation of structurally related serine proteases. Although both chains are required for activation, only the alpha-chain binds Met with high affinity. Recently, we reported that the protease-like HGF beta-chain binds to Met with low affinity (Stamos, J., Lazarus, R. A., Yao, X., Kirchhofer, D., and Wiesmann, C. (2004) EMBO J. 23, 2325-2335). Here we demonstrate that the zymogen-like form of HGF beta also binds Met, albeit with 14-fold lower affinity than the protease-like form, suggesting optimal interactions result from conformational changes upon cleavage of the single-chain form. Extensive mutagenesis of the HGF beta region corresponding to the active site and activation domain of serine proteases showed that 17 of the 38 purified two-chain HGF mutants resulted in impaired cell migration or Met phosphorylation but no loss in Met binding. However, reduced biological activities were well correlated with reduced Met binding of corresponding mutants of HGF beta itself in assays eliminating dominant alpha-chain binding contributions. Moreover, the crystal structure of HGF beta determined at 2.53 A resolution provides a structural context for the mutagenesis data. The functional Met binding site is centered on the "active site region" including "triad" residues Gln(534) [c57], Asp(578) [c102], and Tyr(673) [c195] and neighboring "activation domain" residues Val(692), Pro(693), Gly(694), Arg(695), and Gly(696) [c214-c219]. Together they define a region that bears remarkable resemblance to substrate processing regions of serine proteases. Models of HGF-dependent Met receptor activation are discussed.     

Adipsin, the adipocyte serine protease: gene structure and control of expression by tumor necrosis factor.

We have isolated, mapped and sequenced adipsin, the adipocyte differentiation-dependent serine protease gene. This gene, which is present in a single form in the mouse, spans 1.7 kilobases and contains five exons. While the basic exon structure characteristic of serine protease genes is conserved in adipsin, there is also a fusion of two exons that are separate in other serine proteases. The sequence data also suggests a mechanism of alternative splicing which appears to account for the generation of two adipsin mRNA species differing by only three nucleotides and encoding two different signal peptides. To investigate the control of adipsin expression we have examined the effects of tumor necrosis factor (TNF) on adipocytes. The level of adipsin RNA is dramatically decreased by hormone treatment, but the change occurs more slowly than for other fat cell mRNAs, such as glycerophosphate dehydrogenase. These results show that adipsin is a novel serine protease gene whose expression is regulated by a macrophage-derived factor which modulates expression of other adipocyte-specific RNAs.     

Structure of the human genomic region homologous to the bovine prochymosin-encoding gene

Two human genomic libraries were probed with bovine prochymosin (bPC) cDNA. Recombinant clones covering a genomic region homologous to the entire coding region and flanking sequences of the bPC gene were isolated. Human sequences homologous to exons of the bPC gene are distributed in a DNA fragment of 10 kb. Alignment of the human sequences and the exons of bPC reveals that the human 'exons' 1-3, 5 and 7-9 have sizes identical to the corresponding bovine exons, but a nucleotide (nt) has been deleted in the human exon 4 and two nt in the human exon 6. The aligned human sequence and the coding part of bPC gene share 82% nt homology, the value ranging, in separate exons, from 76 (exon 1) to 84% (exons 5 and 6). 150 bp of 5'-flanking sequence of the human gene has 75% homology to the corresponding region of bPC gene and contains a TATA-box in a similar position. A 1-nt deletion in the human exon 4 would shift the translational reading frame of a putative human PC mRNA relative to bPC mRNA, and result in an in-phase terminator spanning codons 163 and 164 in bPC mRNA. Another terminator in-phase with the amino-acid sequence encoded by the bPC gene occurs in the human exon 5 and the second frameshift mutation in exon 6. Thus, the nt sequence analysis of the human genomic region has revealed the presence of mutations that have rendered it unable to produce a full-length protein homologous to bPC and, therefore, we refer to this gene as a human prochymosin pseudogene (hPC psi). Blot-hybridization analysis of human genomic DNA indicates that hPC psi is a single gene in the human genome.     

Evolution of Thrombin and Other Hemostatic Proteases by Survey of Protochordate, Hemichordate, and Echinoderm Genomes

Protochordate genomes enable a prevalence of hemostasis evolution. Broad searches were performed for homologs of human serine proteases of hemostasis on the genomes of Branchiostoma floridae, Saccoglossus kowalevskii, and Strongylocentrotus purpuratus. Sequences were analyzed by multiple bioinformatic tools. The survey revealed numerous homologous components. Amphioxus was rich in some serine proteases not accompanied by gamma-carboxyglutamic or kringle domains similar more to thrombin than to other coagulation factors. The serine proteases found in amphioxus exhibited the attributes similar to those of thrombin by phylogeny relationships, sequence conservation, gene synteny, spatial structure, and ligand docking. A few plasminogen- and plasminogen activators-like proteases with kringles were also present. Those serine proteases demonstrated the greatest proximity rather to plasminogen or plasminogen activators than to thrombin. Searching for homologs of serine protease hemostatic factors in acorn worm and sea urchin revealed several components similar to those found in amphioxus. Hypothetically, the common ancestor of chordates had three separate serine proteases that evolved independently into immunoglobulin-like and kringle proteases in lancelets, and prothrombin, plasminogen activators, and plasminogen in vertebrates. Ancestral proteases evolved in vertebrates into hemostasis factors after merging the proper N-terminal domains and duplications.     

Molecular evolution and domain structure of plasminogen-related growth factors (HGF/SF and HGF1/MSP).

Plasminogen-related growth factors, a new family of polypeptide growth factors with the basic domain organization and mechanism of activation of the blood proteinase plasminogen, include hepatocyte growth factor/scatter factor (HGF/SF), a potent effector of the growth, movement, and differentiation of epithelia and endothelia, and hepatocyte growth factor-like/macrophage stimulating protein (HGF1/MSP), an effector of macrophage chemotaxis and phagocytosis. Phylogeny of the serine proteinase domains and analysis of intron-exon boundaries and kringle sequences indicate that HGF/SF, HGF1/MSP, plasminogen, and apolipoprotein (a) have evolved from a common ancestral gene that consisted of an N-terminal domain corresponding to plasminogen activation peptide (PAP), 3 copies of the kringle domain, and a serine proteinase domain. Models of the N domains of HGF/SF, HGF1/MSP, and plasminogen, characterized by the presence of 4 conserved Cys residues forming a loop in a loop, have been modeled based on disulfide-bond constraints. There is a distinct pattern of charged and hydrophobic residues in the helix-strand-helix motif proposed for the PAP domain of HGF/SF; these may be important for receptor interaction. Three-dimensional structures of the 4 kringle and the serine proteinase domains of HGF/SF were constructed by comparative modeling using the suite of programs COMPOSER and were energy minimized. Docking of a lysine analogue indicates a putative lysine-binding pocket within kringle 2 (and possibly another in kringle 4). The models suggest a mechanism for the formation of a noncovalent HGF/SF homodimer that may be responsible for the activation of the Met receptor. These data provide evidence for the divergent evolution and structural similarity of plasminogen, HGF/SF, and HGF1/MSP, and highlight a new strategy for growth factor evolution, namely the adaptation of a proteolytic enzyme to a role in receptor activation.     

Structure of the murine immune response I-A beta locus: sequence of the I-A beta gene and an adjacent beta-chain second domain exon

The murine major histocompatibility complex I region encodes two class II antigens, I-A and I-E. From a mouse spleen DNA cosmid library of the b haplotype, we isolated a clone containing the entire I-A beta gene and a separate exon encoding a beta-chain second domain (A beta 2). The A beta gene, encompassing more than 6 kb, is encoded by six exons corresponding to the different domains of the A beta polypeptide. The translated A beta amino acid sequence displays 73% homology to human DC beta chains; homologies to other subsets of human beta chains are lower, establishing that I-A corresponds structurally to DC. The A beta 2 exon is about 20 kb centromeric to the A beta gene. Its translated amino acid sequence includes all the conserved amino acids of other class II beta-chain second domains. It shows about 60% homology to each of three subsets of human beta chains available for comparison, and to the A beta chain. No A beta 2 first domain exon has been detected with A beta or DC beta probes.