ABSTRACT
An imbalance between peptidases and their inhibitors leads to pulmonary disease. Imbalances occur in the adult and the neonate at risk for a specific set of lung pathologies. Serpins (serine peptidase inhibitors) make up the major source of antipeptidase activity in the lung. The purpose of this review is to describe the serpin mechanism of inhibition, their roles in the normal and pathological lung and their potential as therapeutic agents.
Subject(s)
Lung Diseases/metabolism , Serpins/metabolism , Humans , Lung Diseases/enzymology , Risk FactorsABSTRACT
Thirteen monoclonal antibodies directed against squamous cell carcinoma antigens (SCCA1 and SCCA2) were obtained from five international collaborating laboratories participating in the ISOBM TD-10 Workshop. Native and recombinant forms of SCCA were used in a wide variety of approaches to determine the reactivity and specificity of these antibodies. Based on reactivity, the antibodies could be divided into three groups: the SCCA1-reactive group containing those that reacted only with recombinant SCCA1 (rSCCA1) and native SCCA1 (nSCCA1) antigens, the SCCA2-reactive group containing those that reacted only with recombinant SCCA2 (rSCCA2), and the pan-reactive group containing those antibodies that reacted with rSCCA1, nSCCA1, and rSCCA2. Binding to radioiodinated rSCCA1 showed that all reactive antibodies were of a high affinity (K(d) <2 x 10(-9) mol/l). Binding to labelled rSCCA2 demonstrated that five antibodies were of a high affinity (K(d) <2 x 10(-9) mol/l). Antibody reactivity on Western blots was tested with nonreduced and reduced native and recombinant SCCA1 and SCCA2. In general, these findings showed that reduction had little effect on binding to SCCA1, but often a strong effect on the binding to SCCA2. Binding of antibodies to rSCCA1 and rSCCA2 in complexes with cathepsin L and G, respectively, was used to assist in the localization of epitope regions in enzyme-complexed SCCA. Cross-inhibition experiments showed that SCCA1-reactive antibodies represent two different epitope groups, and this is supported by their ability to make SCCA1-specific assays by combining antibodies from the two epitope groups. The SCCA2-reactive group represents two related antibodies and one unique as seen in cross-inhibition, but they do not form successful assay combinations. Classification of the pan-reactive antibodies is more difficult, as some epitope groups differ when results from rSCCA1 are compared with rSCCA2 as the target. However, two antibodies are outstanding, SCC107 and SCC113, as they are high-affinity antibodies which react equally well with free and protease complexes of SCCA1 and SCCA2. The precise location of epitopes was further studied using sequential overlapping peptides and homology modelling. The findings from this workshop strongly indicate that the recombinant antigens (rSCCA1 and rSCCA2) are very similar in epitope structure to the native counterparts in saliva, and squamous epithelium from normal and cancer tissues. Therefore, it is reasonable to conclude that the specificities found are reliable and have application for antibody measurement of all forms of squamous cell carcinoma in serum except SCCA2 in complex with its protease.
Subject(s)
Antibodies, Monoclonal/immunology , Antigens, Neoplasm/immunology , Biomarkers, Tumor/immunology , Carcinoma, Squamous Cell/immunology , Serpins/immunology , Antibodies, Monoclonal/analysis , Antibody Formation , Blotting, Western , Humans , Sensitivity and SpecificityABSTRACT
Serpins are unique among the various types of active site proteinase inhibitors because they covalently trap their targets by undergoing an irreversible conformational rearrangement. Members of the serpin superfamily are present in the three major domains of life (Bacteria, Archaea and Eukarya) as well as several eukaryotic viruses. The human genome encodes for at least 35 members that segregate evolutionarily into nine (A-I) distinct clades. Most of the human serpins are secreted and circulate in the bloodstream where they reside at critical checkpoints intersecting self-perpetuating proteolytic cascades such as those of the clotting, thrombolytic and complement systems. Unlike these circulating serpins, the clade B serpins (ov-serpins) lack signal peptides and reside primarily within cells. Most of the human clade B serpins inhibit serine and/or papain-like cysteine proteinases and protect cells from exogenous and endogenous proteinase-mediated injury. Moreover, as sequencing projects expand to the genomes of other species, it has become apparent that intracellular serpins belonging to distinct phylogenic clades are also present in the three major domains of life. As some of these serpins also guard cells against the deleterious effects of promiscuous proteolytic activity, we propose that this cytoprotective function, along with similarities in structure are common features of a cohort of intracellular serpin clades from a wide variety of species.
Subject(s)
Serine Proteinase Inhibitors/metabolism , Serpins/metabolism , Amino Acid Sequence , Animals , Evolution, Molecular , Gene Expression Regulation , Humans , Models, Molecular , Molecular Sequence Data , Phylogeny , Protein Structure, Tertiary , Sequence Alignment , Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/classification , Serine Proteinase Inhibitors/genetics , Serpins/chemistry , Serpins/classification , Serpins/geneticsABSTRACT
Members of the human serpin family regulate a diverse array of serine and cysteine proteinases associated with essential biological processes such as fibrinolysis, coagulation, inflammation, cell mobility, cellular differentiation, and apoptosis. Most serpins are secreted and attain physiologic concentrations in the blood and extracellular fluids. However, a subset of the serpin superfamily, the ov-serpins, also resides intracellularly. Using high throughput genomic sequence, we identified a novel member of the human ov-serpin gene family, SERPINB12. The gene mapped to the ov-serpin cluster at 18q21 and resided between SERPINB5 (maspin) and SERPINB13 (headpin). The presence of SERPINB12 in silico was confirmed by cDNA cloning. Expression studies showed that SERPINB12 was expressed in many tissues, including brain, bone marrow, lymph node, heart, lung, liver, pancreas, testis, ovary, and intestines. Based on the presence of Arg and Ser at the reactive center of the RSL, SERPINB12 appeared to be an inhibitor of trypsin-like serine proteinases. This hypothesis was confirmed because recombinant SERPINB12 inhibited human trypsin and plasmin but not thrombin, coagulation factor Xa, or urokinase-type plasminogen activator. The second-order rate constants for the inhibitory reactions were 2.5 +/- 1.6 x 10(5) and 1.6 +/- 0.2 x 10(4) M(-1) S(-1), respectively. These data show that SERPINB12 encodes for a new functional member of the human ov-serpin family.
Subject(s)
Serine Proteinase Inhibitors/metabolism , Serpins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Humans , Molecular Sequence Data , Multigene Family , Protein Denaturation , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/genetics , Serpins/chemistry , Serpins/genetics , Tissue DistributionABSTRACT
Squamous cell carcinoma (SCC) antigen has been used for the management of SCC arising in various cites including head and neck region. However, the true mechanism of the elevation of this protein in the serum of patients with SCC is still unknown. SCC antigen belongs to the superfamily of serine protease inhibitors. Recently, molecular studies show that serum SCC antigen is transcribed by two nearly identical genes (SCCA1 and SCCA2), and is mainly produced by SCCA1. The objective of this study is to clarify the mechanism of the elevation of SCC antigen in oral tongue SCC patients and to identify cells histologically, which are responsible for serum SCC antigen production. In this study, we examined SCCA1 expression in a series of four head and neck SCC (HNSCC) cell lines, and found that all expressed equal to low SCCA1 protein as compared with the normal human oral keratinocyte. Using the double immunohistochemical technique to examine the expression pattern of SCCA1 in 86 cases of oral tongue squamous cell carcinoma, SCCA1 immunostaining was observed in the cytoplasm of cancer cells and T-lymphocytes peripheral to cancer cells. We also compared the clinicopathological features including serum SCC antigen level of the oral tongue SCC cases with the immunohistochemical SCCA1 expression pattern, and found that elevated serum SCC antigen level was significantly correlated with SCCA1 expression not in cancer cells, but in T-lymphocytes peripheral to cancer cells. These results suggest that T-lymphocytes peripheral to cancer cells may be responsible for serum SCC antigen production in HNSCC patients.
Subject(s)
Antigens, Neoplasm/biosynthesis , Antigens, Neoplasm/blood , Carcinoma, Squamous Cell/blood , Serpins , T-Lymphocytes/metabolism , Tongue Neoplasms/blood , Adult , Aged , Aged, 80 and over , Blotting, Western , Carcinoma, Squamous Cell/mortality , Female , Humans , Immunohistochemistry , Lymph Nodes/metabolism , Male , Middle Aged , Survival Rate , Tongue Neoplasms/mortality , Tumor Cells, CulturedABSTRACT
Squamous cell carcinoma (SCC) antigen (SCCA), a member of the ovalbumin serine proteinase inhibitor family, serves as a circulating marker of squamous cell carcinoma (SC). One of the SCCAs, SCCA1, has been suggested to play a role in the attenuation of apoptosis in vitro and in the augmentation of tumor growth in vivo. In the present study, the infection of a SCC cell line (SKG IIIa) with recombinant retrovirus that expressed the antisense SCCA mRNA suppressed expression of SCCA in vitro. Local administration of this retrovirus into tumors by inoculation in nude mice suppressed tumor growth. Treatment of tumor tissue in vivo is also associated with increased numbers of apoptotic tumor cells and large mononuclear cells in the tumor. To test the possible role of SCCA in the infiltration of large mononuclear cells, we analyzed the effect of SCCA1 on migration of natural killer (NK) cells induced by monocyte-chemoattractant protein-1 in vitro. SCCA1 suppressed migration of NK cells completely, and this inhibitory effect was lost by mutation of the reactive site loop of SCCA1. These results suggest that antisense SCCA may suppress the growth of SCC in vivo not only by the augmentation of intracellular apoptosis but also by the increased infiltration of NK cells into the tumor.
Subject(s)
Antigens, Neoplasm/biosynthesis , Carcinoma, Squamous Cell/pathology , Killer Cells, Natural/pathology , Oligonucleotides, Antisense/pharmacology , Serpins/biosynthesis , 3T3 Cells , Animals , Antigens, Neoplasm/genetics , Carcinoma, Squamous Cell/drug therapy , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/metabolism , Cell Division , Cell Movement/drug effects , Humans , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Oligonucleotides, Antisense/genetics , Serpins/genetics , Transduction, GeneticABSTRACT
Despite the relatively large cloning capacity of YACs, many genomic regions or individual genes are not cloned intact, but are represented as a collection of overlapping clones or contigs. Fortunately, the relatively high frequency and fidelity of homologous recombination in Saccharomyces cerevisiae can be used to reconstruct intact genes within a single clone by splicing together overlapping DNA segments. This unit describes two protocols for carrying out such homologous recombination; one relies on the meiotic phase of the yeast cycle, while the other utilizes the mitotic phase of the yeast life cycle. Despite the relatively large cloning capacity of YACs, many genomic regions or individual genes are not cloned intact.
Subject(s)
Chromosomes, Artificial, Yeast/genetics , Recombination, Genetic , Diploidy , Genetic Techniques , Genetics, Medical , Genotype , Humans , Meiosis/genetics , Mitosis/genetics , Polymerase Chain Reaction , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/geneticsABSTRACT
Headpin is a novel serine proteinase inhibitor (serpin) that is down-regulated in squamous cell carcinoma of the oral cavity and in squamous cell carcinoma cell lines of the head and neck. Using a panel of 18q21.3 YAC clones, we mapped and cloned the HEADPIN gene. The gene spans 10 kb and is composed of eight exons and seven introns. The genomic structure is identical with some other ovalbumin serpins (ov-serpins) in terms of the numbers, position and phasing of the intron/exon boundaries. HEADPIN was mapped within the serpin cluster in 18q21.3 between MASPIN and SCCA2 as follows: cen-MASPIN-HEADPIN-SCCA2-SCCA1-tel. The transcription start site was determined and the promoter activity of the 5'-flanking region was analyzed. Luciferase promoter assays in HaCaT cells showed that the -432 to -144 nucleotide region has functional promoter activity. The activity of the promoter/enhancer was not observed in head and neck cancer cell lines TU167 and UMSCC1 which lack headpin expression. These data suggest that the differential expression of headpin in normal and carcinoma-derived cells is regulated at the transcriptional level. Understanding the genomic organization and transcriptional regulation of the ov-serpins clustered within 18q21. 3 provides a critical framework for assessing their potential role in cancer.
Subject(s)
Gene Expression Regulation, Neoplastic , Promoter Regions, Genetic/genetics , Serpins/genetics , Base Sequence , Chromosome Mapping , Chromosomes, Human, Pair 18 , Cloning, Molecular , DNA/analysis , Down-Regulation , Exons , Genome, Human , Head and Neck Neoplasms/genetics , Humans , Introns , Molecular Sequence Data , TATA BoxABSTRACT
An elevation in the circulating level of the squamous-cell carcinoma antigen (SCCA) can be a poor prognostic indicator in certain types of squamous-cell cancers. Total SCCA in the circulation comprises 2 nearly identical, approximately 45 kDa proteins, SCCA1 and SCCA2. Both proteins are members of the high-molecular weight serine proteinase inhibitor (serpin) family with SCCA1 paradoxically inhibiting lysosomal cysteine proteinases and SCCA2 inhibiting chymotrypsin-like serine proteinases. Although SCCA1 and SCCA2 are detected in the cytoplasm of normal squamous epithelial cells, neither serpin is detected normally in the serum. Thus, their presence in the circulation at relatively high concentrations suggests that malignant epithelial cells are re-directing serpin activity to the fluid phase via an active secretory process. Because serpins typically inhibit their targets by binding at 1:1 stoichiometry, a change in the distribution pattern of SCCA1 and SCCA2 (i.e., intracellular to extracellular) could indicate the need of tumor cells to neutralize harmful extracellular proteinases. The purpose of our study was to determine experimentally the fate of SCCA1 and SCCA2 in squamous carcinoma cells. Using subcellular fractionation, SCCA-green fluorescent fusion protein expression and confocal microscopy, SCCA1 and SCCA2 were found exclusively in the cytosol and were not associated with nuclei, mitochondria, lysosomes, microtubules, actin or the Golgi. In contrast to previous reports, metabolic labeling and pulse-chase experiments showed that neither non-stimulated nor TNFalpha/PMA-stimulated squamous carcinoma cells appreciably secreted these ov-serpins into the medium. Collectively, these data suggest that the major site of SCCA1 and SCCA2 inhibitory activity remains within the cytosol and that their presence in the sera of patients with advanced squamous-cell carcinomas may be due to their passive release into the circulation.
Subject(s)
Antigens, Neoplasm/metabolism , Biomarkers, Tumor/metabolism , Serpins/metabolism , Amino Acid Sequence , Animals , Antigens, Neoplasm/blood , Biomarkers, Tumor/blood , COS Cells , Cytosol/metabolism , Endopeptidases/metabolism , Female , Head and Neck Neoplasms/blood , Head and Neck Neoplasms/metabolism , Humans , Molecular Sequence Data , Protease Inhibitors/blood , Protease Inhibitors/metabolism , Sequence Homology, Amino Acid , Serpins/blood , Subcellular Fractions/metabolism , Tetradecanoylphorbol Acetate/pharmacology , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/pharmacology , Uterine Cervical Neoplasms/blood , Uterine Cervical Neoplasms/metabolismABSTRACT
The human squamous cell carcinoma antigens (SCCA) 1 and 2 are members of the serpin family that are 92% identical in their amino acid sequence. Despite this similarity, they inhibit distinct classes of proteinases. SCCA1 neutralizes the papain-like cysteine proteinases, cathepsins (cat) S, L, and K; and SCCA2 inhibits the chymotrypsin-like serine proteinases, catG and human mast cell chymase. SCCA2 also can inhibit catS, as well as other papain-like cysteine proteinases, albeit at a rate 50-fold less than that of SCCA1. Analysis of the mechanism of inhibition by SCCA1 revealed that the reactive site loop (RSL) is important for cysteine proteinase inhibition. The inhibition of catS by a mutant SCCA2 containing the RSL of SCCA1 is comparable to that of wild-type SCCA1. This finding suggested that there were no motifs outside and only eight residues within the RSL that were directing catS-specific inhibition. The purpose of this study was to determine which of these residues might account for the marked difference in the ability of SCCA1 and SCCA2 to inhibit papain-like cysteine proteinases. SCCA2 molecules containing different RSL mutations showed that no single amino acid substitution could convert SCCA2 into a more potent cysteine proteinase inhibitor. Rather, different combinations of mutations led to incremental increases in catS inhibitory activity with residues in four positions (P1, P3', P4', and P11') accounting for 80% of the difference in activity between SCCA1 and SCCA2. Interestingly, the RSL cleavage site differed between wild-type SCCA2 and this mutant. Moreover, these data established the importance of a Pro residue in the P3' position for efficient inhibition of catS by both wild-type SCCA1 and mutated SCCA2. Molecular modeling studies suggested that this residue might facilitate positioning of the RSL within the active site of the cysteine proteinase.
Subject(s)
Antigens, Neoplasm/chemistry , Serpins/chemistry , Amino Acid Sequence , Antigens, Neoplasm/genetics , Antigens, Neoplasm/pharmacology , Binding Sites , Cathepsins/antagonists & inhibitors , Elastin/metabolism , Enzyme Stability , Humans , Kinetics , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Papain/metabolism , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Proline/genetics , Proline/metabolism , Sequence Alignment , Serpins/geneticsABSTRACT
The squamous cell carcinoma antigen (SCCA) serves as a serologic marker for advanced squamous cell carcinomas (SCC) of the uterine cervix, lung, esophagus, head and neck and vulva. Elevations in serum levels of SCCA following treatment for SCC correlate with tumor relapse or metastasis. Recent molecular studies show that SCCA is transcribed by two nearly identical genes (SCCA1 and SCCA2) that encode for members of the high molecular weight serine proteinase inhibitor (serpin) family. Despite a high degree of similarity in their amino acid sequences, SCCA1 and SCCA2 have distinct biochemical properties: SCCA1 is an inhibitor of papain like cysteine proteinases, such as cathepsins (cat) L, S and K, whereas SCCA2 inhibits chymotrypsin-like serine proteinases, catG and mast cell chymase. In this paper, we report the generation and characterization of anti-SCCA1 and anti-SCCA2 specific monoclonal antibodies (MAbs). Using these MAbs, we developed an enzyme-linked immunoassay (ELISA) that discriminated between SCCA1 and SCCA2 without any cross-reaction. This assay measured both the native and complexed forms of SCCA1 and SCCA2. The sensitivity of detection of SCCA1 and SCCA2 assays were 0.17 ngml(-1) and 0.19 ngml(-1), respectively. Mean inter- and intra-assay coefficients of variation were 12.1% and 9.9% for SCCA1 assay and 12% and 8.8% for SCCA2 assay, respectively. Recovery and parallellism studies indicated that SCCA1 and SCCA2 were detected in the plasma and amniotic fluids without any major interference by the biologic fluid components. This assay provides a simple and accurate procedure for the quantitation of total SCCA1 and SCCA2.
Subject(s)
Antibodies, Monoclonal/immunology , Antigens, Neoplasm/immunology , Biomarkers, Tumor/immunology , Enzyme-Linked Immunosorbent Assay/methods , Serpins , Animals , Antibody Specificity , Antigens, Neoplasm/blood , Biomarkers, Tumor/blood , Mice , Mice, Inbred BALB C , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
The serpins SCCA1 and SCCA2 are highly expressed in the epithelium of the conducting airways, a common site of infection by group C adenoviruses, such as human adenovirus type 2 (Ad2). Based on the common location we examined a possible interaction between them. In vitro experiments with recombinant proteins showed that SCCA1 inhibited the viral protease in a dose-dependent manner. Both serpins were cleaved in a manner consistent with hydrolysis within their reactive site loop, without the formation of an SDS-resistant complex, as in the case of papain. Infection of SCCA1-expressing cells did not result in the cleavage of SCCA1, nor was the yield of infectious virus affected as compared to SCCA1-negative parental cells. This may be due to differential localization, the serpin being cytoplasmic and viral protease being nuclear. Surprisingly, however, virus infection, which tends to inhibit host protein synthesis, caused a significant increase in SCCA1 expression well into the late phase of infection.
Subject(s)
Adenoviruses, Human/enzymology , Antigens, Neoplasm/metabolism , Cysteine Endopeptidases/metabolism , Serpins , Adenocarcinoma , Adenoviruses, Human/physiology , Antigens, Neoplasm/genetics , Breast Neoplasms , Female , Humans , Laryngeal Neoplasms , Recombinant Fusion Proteins/metabolism , Tumor Cells, CulturedABSTRACT
Squamous cell carcinoma antigen (SCCA) serves as a serological marker for advanced squamous cell carcinomas (SCCs) and as an indicator of therapeutic response. Recent molecular studies show that the SCCA is transcribed by two almost identical tandemly arrayed genes, SCCA1 and SCCA2. These genes are members of the high molecular weight serine proteinase inhibitor (serpin) superfamily. Although SCCA1 and SCCA2 are 92% identical at the amino acid level, they have distinct biochemical properties. Paradoxically, SCCA1 is an inhibitor of papain-like cysteine proteinases, such as cathepsins L, S, and K, whereas SCCA2 inhibits chymotrypsin-like serine proteinases, cathepsin G, and mast cell chymase. Using a new set of discriminatory monoclonal antibodies (MAbs) and polymerase chain reaction (PCR) assay, we showed that SCCA1 and SCCA2 were co-expressed in the suprabasal layers of the stratified squamous epithelium of the tongue, tonsil, esophagus, uterine cervix and vagina, Hassall's corpuscles of the thymus, and some areas of the skin. SCCA1 and SCCA2 also were detected in the pseudo-stratified columnar epithelium of the conducting airways. Examination of squamous cell carcinomas of the lung and head and neck showed that SCCA1 and SCCA2 were co-expressed in moderately and well-differentiated tumors. Moreover, there was no differential expression between these SCCA "isoforms" in normal or malignant tissues. In contrast to previous studies, these data indicated that the expression of SCCA1 and SCCA2 was not restricted to the squamous epithelium and that these serpins may coordinately regulate cysteine and serine proteinase activity in both normal and transformed tissues.
Subject(s)
Antigens, Neoplasm/isolation & purification , Biomarkers, Tumor/isolation & purification , Carcinoma, Squamous Cell/chemistry , Serpins/isolation & purification , Antibodies, Monoclonal , Antibody Specificity , Antigens, Neoplasm/genetics , Biomarkers, Tumor/genetics , Carcinoma, Squamous Cell/pathology , Epithelium/chemistry , Female , Humans , Pregnancy , Protein Isoforms , Reverse Transcriptase Polymerase Chain Reaction , Serpins/genetics , Tissue DistributionABSTRACT
Numerous positional cloning projects directed at isolating genes responsible for the myriads of observed developmental defects in the zebrafish are anticipated in the very near future. In this chapter, we have reviewed the YAC, BAC, and PAC large-insert genomic resources available to the zebrafish community. We have discussed how these resources are screened and used in a positional cloning scheme and have pointed out frequently formidable logistical considerations in the approach. Despite being extremely tedious, positional cloning projects in the zebrafish will be comparatively easier to accomplish than in human and mouse, because of unique biological advantages of the zebrafish system. Moreover, the ease and speed at which genes are identified and cloned should rapidly increase as more mapping reagents and information become available, thereby paving the way for meaningful biological studies.
Subject(s)
Chromosomes, Artificial, Yeast , Genomic Library , Zebrafish/genetics , Animals , Bacteria , Cloning, Molecular , Genetic Testing/methods , Humans , Internet , MiceABSTRACT
The 18q-syndrome is representative of a group of terminal deficiency or macrodeletion syndromes characterized by mental retardation and congenital malformations. To gain insight into the mechanism of chromosomal loss and stabilization in these disorders, we cloned a putative terminal deletion breakpoint from an 18q-syndrome patient. The 18q21.3 breakpoint occurred between two nearly identical serine protease inhibitor (serpin) genes, SCCA1 and SCCA2. Although cytogenetic studies suggested that this chromosomal aberration was formed by a simple terminal deletion, DNA sequence analysis, pulsed-field gel electrophoresis and fluorescence in situ hybridization showed that the breakpoint was contiguous with a 35 bp filler sequence followed by a satellite III DNA-containing telomeric fragment of 475-1000 kb. This type of satellite III DNA sequence was not detected on the normal chromosome 18, but was highly homologous with types of satellite III DNA sequences normally located on the short arms (p11) of the acrocentric chromosomes and other heterochromatic regions. This DNA sequence analysis suggested that the terminal deficiency in this 18q-syndrome patient arose via illegitimate (non-homologous) recombination. Moreover, these data raise the possibility that a subset of chromosomal aberrations appearing cytogenetically and molecularly as simple terminal truncations or deletions are caused by small (<1000 kb) cryptic rearrangements.
Subject(s)
Antigens, Neoplasm/genetics , Chromosome Breakage/genetics , Chromosome Deletion , Chromosomes, Human, Pair 18/genetics , Serpins/genetics , Abnormalities, Multiple/genetics , Base Sequence , Cell Line , Chromosome Mapping , DNA, Satellite/genetics , Gene Rearrangement , Humans , In Situ Hybridization, Fluorescence , Intellectual Disability/genetics , Molecular Sequence Data , Repetitive Sequences, Nucleic Acid , Syndrome , Telomere/geneticsABSTRACT
The high-molecular-weight serine proteinase inhibitors (serpins) are restricted, generally, to inhibiting proteinases of the serine mechanistic class. However, the viral serpin, cytokine response modifier A, and the human serpins, antichymotrypsin and squamous cell carcinoma antigen 1 (SCCA1), inhibit different members of the cysteine proteinase class. Although serpins employ a mobile reactive site loop (RSL) to bait and trap their target serine proteinases, the mechanism by which they inactivate cysteine proteinases is unknown. Our previous studies suggest that SCCA1 inhibits papain-like cysteine proteinases in a manner similar to that observed for serpin-serine proteinase interactions. However, we could not preclude the possibility of an inhibitory mechanism that did not require the serpin RSL. To test this possibility, we employed site-directed mutagenesis to alter the different residues within the RSL. Mutations to either the hinge or the variable region of the RSL abolished inhibitory activity. Moreover, RSL swaps between SCCA1 and the nearly identical serpin, SCCA2 (an inhibitor of chymotrypsin-like serine proteinases), reversed their target specificities. Thus, there were no unique motifs within the framework of SCCA1 that independently accounted for cysteine proteinase inhibitory activity. Collectively, these data suggested that the sequence and mobility of the RSL of SCCA1 are essential for cysteine proteinase inhibition and that serpins are likely to utilize a common RSL-dependent mechanism to inhibit both serine and cysteine proteinases.
Subject(s)
Antigens, Neoplasm/chemistry , Serine Proteinase Inhibitors/chemistry , Serpins/chemistry , Amino Acid Sequence , Animals , Antigens, Neoplasm/genetics , Antigens, Neoplasm/metabolism , Binding Sites , Escherichia coli , Humans , Molecular Sequence Data , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/genetics , Serine Proteinase Inhibitors/metabolism , Serpins/genetics , Serpins/metabolism , Structure-Activity RelationshipABSTRACT
The genes for the squamous cell carcinoma antigen (SCCA) were found flanking a deletion breakpoint from a patient with the 18q-syndrome. The genes are <10 kb apart, tandemly arrayed in a head-to-tail fashion, and approximately 10 kb in size. Both genes also contain 8 exons and identical intron-exon boundaries. The cDNAs encode for proteins that are 92% identical and 95% similar. Amino acid comparisons show that SCCA1 and SCCA2 are members of the high-molecular weight serine proteinase inhibitor (serpin) family. Physical mapping studies show that the genes reside within the 500-kb region of 18q21.3 that contains at least four other serpin genes. The gene order is cen-maspin (PI5), SCCA2, SCCA1, PAI2, bomapin (PI10), PI8-tel. Biochemical analysis of recombinant SCCA1 and SCCA2 proteins shows that SCCA1 is a potent cross-class inhibitor of papain-like cysteine proteinases such as cathepsins L, S and K, whereas SCCA2 is an inhibitor of chymotrypsin-like serine proteinases such as cathepsin G and mast cell chymase. These findings suggest that SCCA1 and SCCA2 are capable of regulating proteolytic events involved in both normal (e.g., tissue remodeling, protein processing) and pathologic processes (e.g., tumor progression).
Subject(s)
Antigens, Neoplasm/genetics , Biomarkers, Tumor/genetics , Chromosomes, Human, Pair 18/genetics , Serpins/genetics , Amino Acid Sequence , Base Sequence , Chromosome Mapping , Humans , Molecular Sequence Data , Protease InhibitorsABSTRACT
Squamous cell carcinoma antigens (SCCA) 1 and 2 are inhibitory members of the high-molecular-weight serine proteinase inhibitor (serpin) family. The biological functions of SCCA1 and 2 are unknown. One approach to determining the function of human proteins is to study orthologs in other species, such as the mouse. The purpose of this study was to determine whether orthologs to human SCCA1 or 2 exist in the mouse. We report the identification and characterization of a novel serpin, sqn5 (now designated Scca2). Comparative amino acid sequence analysis suggests that Scca2 is a member of the ov-serpin subfamily of serpins with highest homology to SCCA1 and SCCA2. Fluorescence in situ hybridization revealed that the Scca2 mapped near Bcl2 on mouse chromosome 1. This region is syntenic with the human locus for SCCA1 and SCCA2 on 18q21.3. The tissue expression patterns as determined by RT-PCR showed a restricted distribution. Scca2 was detected in the lung, thymus, skin, and uterus, as are SCCA1 and SCCA2. Unlike the SCCAs, however, Scca2 was detected also in the gastrointestinal tract. Enzyme-inhibition assays using a GST-SCCA2 fusion protein revealed that SCCA2 inhibited chymotrypsin-like serine proteinases, but not papain-like cysteine proteinases. SCCA2 inhibited CTSG at 1:1 stoichiometry and with a second-order rate constant of kass = 1.7 x 10(5) M-1 s-1. SCCA2 also inhibited human mast cell chymase but the stoichiometry was 2:1, and the second-order rate constant was kass = 0.9 x 10(4) M-1 s-1. This inhibitory profile is identical to that observed for human SCCA2. Based on these findings, Scca2 appears to be the murine ortholog of human SCCA2.
Subject(s)
Antigens, Neoplasm/genetics , Chromosomes, Human, Pair 1/genetics , Serine Proteinase Inhibitors/genetics , Serpins , Amino Acid Sequence , Animals , Base Sequence , Chromosome Mapping , Chymases , Cloning, Molecular , Conserved Sequence/genetics , Humans , In Situ Hybridization, Fluorescence , Kinetics , Mice , Molecular Sequence Data , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , Recombinant Fusion Proteins/genetics , Restriction Mapping , Sequence Alignment , Sequence Analysis, DNA , Serine Endopeptidases/metabolismABSTRACT
The breakpoint of the 18q21 translocation of B-cell-non-Hodgkin's lymphoma (NHL) cell line Karpas1106P was delineated by fluorescence in situ hybridization (FISH). Karpas1106P was derived from mediastinal lymphoblastic B-cell lymphoma and exhibited the immunophenotype characteristic of marginal-zone B-cell lymphoma (MZL): smIg+, pan-B antigen+, CD5-, CD10- and CD23-. The original G-banded karyotype showed a complex translocation containing t(X;18;13)(q28;q21;q12.1). Double-color FISH (DCFISH) with whole-chromosome-painting (WCP) probes for chromosomes X, 13 and 18, and 18q-specific yeast artificial chromosome (YAC) clones defined t(X;18;13) as ider(X)t(X;18; 13)(q28;q 12.3q21.1;q12.1). The immunoglobulin-heavy-chain (IgH) gene was not involved in the chromosomal translocation as detected by DCFISH with VH and Cgamma probes. By using contiguous YAC clones mapped from 18q12.3 to q21.1, we identified a YAC clone y852H2 with its breakpoint at 18q21.1. In Karpas1106P, the distal part of chromosome 18 from the breakpoint (18q21.1-qter) was deleted, showing loss of heterozygosity of this region. In addition, the chromosomal segment 18q21.1 was duplicated and inserted to ider(X)t(X;18; 13) between Xq28 and 13q12.1 with maintaining its original orientation. The DNA sequence of the breakpoint region contained in y852H2 can serve as a candidate locus for further molecular dissection to identify the causative gene of MZL.