Signal Peptidase Cleavage Site Research Articles

Determinants of Topogenesis and Glycosylation of Type II Membrane Proteins: ANALYSIS OF Na,K-ATPase β1 AND β3SUBUNITS BY GLYCOSYLATION MAPPING

The structural and molecular determinants that govern the correct membrane insertion and folding of membrane proteins are still ill-defined. By following the addition of sugar chains to engineered glycosylation sites (glycosylation mapping) in Na,K-ATPase beta isoforms expressed in vitro and in Xenopus oocytes, in combination with biochemical techniques, we have defined the C-terminal end of the transmembrane domain of these type II proteins. N-terminal truncation and the removal of a single charged residue at the N-terminal start of the putative transmembrane domain influence the proper positioning of the transmembrane domain in the membrane as reflected by a repositioning of the transmembrane domain, the exposure of a putative cryptic signal peptidase cleavage site, and the production of protein species unable to insert into the membrane. Glycosylation mapping in vivo revealed that the degree of glycosylation at acceptor sites located close to the membrane increases with the time proteins spend in the endoplasmic reticulum. Furthermore, core sugars added to such acceptor sites cannot be processed to fully glycosylated species even when the protein is transported to the cell surface. Thus, the glycosylation mapping strategy applied in intact cells is a useful tool for the study of determinants for the correct membrane insertion of type II and probably other membrane proteins, as well as for the processing of sugar chains in glycoproteins.

Identification of the gene encoding BmpB, a 30 kDa outer envelope lipoprotein of Brachyspira (Serpulina) hyodysenteriae, and immunogenicity of recombinant BmpB in mice and pigs.

A gene encoding a 30kDa outer envelope protein of the intestinal spirochaete Brachyspira (Serpulina) hyodysenteriae, was cloned and expressed in Escherichia coli strain XLOLR. Five phagemids containing DNA inserts encoding the protein were established and one clone (pSHA) was sequenced. An 816bp hypothetical open reading frame (ORF) was identified, with a potential ribosome binding site (AGGAG), and putative -10 (TATAAT) and -35 (TTGAAA) promoter regions upstream from the ATG start of the ORF. A 12bp inverted repeat sequence, possibly serving as a transcription terminator, was identified downstream from the TAA stop codon. Analysis of the amino acid sequence identified a 19 residue hydrophobic signal peptide, incorporating a potential signal peptidase cleavage site and membrane lipoprotein lipid attachment site. Further analysis of the amino acid usage of this lipoprotein, designated BmpB, showed its possible outer membrane localisation. Comparison of the gene encoding the lipoprotein, bmpB, with GenBank nucleotide sequences showed that it has homology with the gene (plp3) encoding Plp3, an outer membrane lipoprotein of Pasteurella haemolytica (54% identity in 735bp). Comparison of the deduced amino acid sequence with the SWISS-PROT amino acid database revealed greatest homology with the outer membrane lipoproteins (Plp1, 2, 3) of P. haemolytica (34% identity in 242 aa, 37% identity in 250 aa, and 39% identity in 272 aa, respectively), and lipoproteins (rcsF and lipoprotein-28) of E. coli (40% identity in 267 aa and 36% identity in 263 aa, respectively). Three of the recombinant E. coli clones (pSHA, pSHD, and pSHE) were formalinised and used to immunise mice. A bacterin preparation of one recombinant E. coli clone (pSHA) was used to immunise pigs. Sera from these mice and pigs recognised the 30kDa lipoprotein in outer membrane preparations of B. hyodysenteriae, indicating the immunogenicity of recombinant BmpB. Sera from pigs naturally infected with B. hyodysenteriae also reacted with recombinant BmpB expressed in E. coli.

Isolation, sequencing and expression of Bartonella henselae omp43 and predicted membrane topology of the deduced protein

The infection of and interaction of human endothelial cells with Bartonella henselae is one of the most interesting aspects of Bartonella -associated disease. The gene encoding the 43 kDa B. henselae outer membrane protein (Omp43) that binds endothelial cells was cloned and sequenced. Sequence analysis revealed an open reading frame of 1206 nucleotides coding for a protein of 402 amino acids. Analysis of the deduced amino acid sequence shows 38% identity over the entire sequence to the Brucella spp. In addition to this Omp2b porin also shows a signal sequence and peptidase cleavage site. Cleavage of the signal peptide results in a mature 380 amino acid polypeptide with a predicted molecular weight of 42 kDa. Omp43 was expressed in Escherichia coli as a fusion protein. Purified recombinant Omp43 at concentrations of 11 and 2.75 μg/ml bound to intact human umbilical vein endothelial cells. Membrane topology analysis predicts that Omp43 exists as a 16 stranded beta barrel protein, similar to that predicted for the Omp2b Brucella abortus porin. Characterization and expression of the gene encoding Omp43 should provide a tool for further investigation of the role of adherence to endothelial cells in the pathogenesis of B. henselae.

Two rat surfactant protein A isoforms arise by a novel mechanism that includes alternative translation initiation.

A single gene for rat surfactant protein A (SP-A) encodes two isoforms that are distinguished by an isoleucine-lysine-cysteine (IKC) N-terminal extension (SP-A and IKC-SP-A). Available evidence suggests that the variants are generated by alternative signal peptidase cleavage of the nascent polypeptide at a primary site (Cys(-)(1)-Asn(1)) and a secondary site (Gly(-)(4)-Ile(-)(3)). In this study, we used site-directed mutagenesis and heterologous expression in vitro and in insect cells to the examine mechanisms that may lead to alternative signal peptidase cleavage including alternative translation initiation at two in-frame AUGs (Met(-)(30) and Met(-)(20)), a suboptimal context for hydrolysis at the primary cleavage site, or cotranslational protein modifications that expose an otherwise cryptic secondary cleavage site. In vitro translation of a rat cDNA for SP-A resulted in both 28 and 29 kDa primary translation products on SDS-PAGE analysis, while translation of cDNAs encoding Met-30Ala and Met-20Ala mutations resulted in only the single 28 and 29 kDa molecular mass species, respectively. These data are consistent with translation initiation at both Met(-)(30) and Met(-)(20) during in vitro synthesis of SP-A. The Met-30Ala mutation reduced expression of the longer isoform in insect cells, indicating that the Met(-)(30) site also contributes to eucaryotic protein expression. Forcing translation initiation at Met(-)(30) by optimizing the Kozak consensus sequence surrounding that codon or by mutating the Met(-)(20) codon resulted in preferential expression of the longer SP-A isoform but reduced overall expression of the protein almost 10-fold. Both isoforms were generated to some degree whether translation was initiated at the codon for Met(-)(30) or Met(-)(20), indicating that the site of translation initiation is not the sole determinant of isoform generation and suggesting that either the context of the primary cleavage site is suboptimal or that cotranslational modifications affect cleavage. Preventing N-terminal glycosylation at Asn(1) did not affect the site of signal peptidase cleavage. Disruption of interchain disulfide formation at Cys(-)(1) by substitution with serine markedly enhanced cleavage at the Gly(-)(4)-Ile(-)(3) bond, but substitution with alanine enhanced cleavage at the Cys(-)(1)-Asn(1) bond. We conclude that rat SP-A isoforms arise by a novel mechanism that includes both alternative translation initiation at two in-frame AUGs and a suboptimal context for signal peptidase hydrolysis at the primary cleavage site.

The leptospiral major outer membrane protein LipL32 is a lipoprotein expressed during mammalian infection.

We report the cloning of the gene encoding the 32-kDa lipoprotein, designated LipL32, the most prominent protein in the leptospiral protein profile. We obtained the N-terminal amino acid sequence of a staphylococcal V8 proteolytic-digest fragment to design an oligonucleotide probe. A Lambda-Zap II library containing EcoRI fragments of Leptospira kirschneri DNA was screened, and a 5.0-kb DNA fragment which contained the entire structural lipL32 gene was identified. Several lines of evidence indicate that LipL32 is lipid modified in a manner similar to that of other procaryotic lipoproteins. The deduced amino acid sequence of LipL32 would encode a 272-amino-acid polypeptide with a 19-amino-acid signal peptide, followed by a lipoprotein signal peptidase cleavage site. LipL32 is intrinsically labeled during incubation of L. kirschneri in media containing [(3)H]palmitate. The linkage of palmitate and the amino-terminal cysteine of LipL32 is acid labile. LipL32 is completely solubilized by Triton X-114 extraction of L. kirschneri; phase separation results in partitioning of LipL32 exclusively into the hydrophobic, detergent phase, indicating that it is a component of the leptospiral outer membrane. CaCl(2) (20 mM) must be present during phase separation for recovery of LipL32. LipL32 is expressed not only during cultivation but also during mammalian infection. Immunohistochemistry demonstrated intense LipL32 reactivity with L. kirschneri infecting proximal tubules of hamster kidneys. LipL32 is also a prominent immunogen during human leptospirosis. The sequence and expression of LipL32 is highly conserved among pathogenic Leptospira species. These findings indicate that LipL32 may be important in the pathogenesis, diagnosis, and prevention of leptospirosis.

SALP16, a gene induced in Ixodes scapularis salivary glands during tick feeding.

Guinea pigs infested with Ixodes scapularis acquire antibody-mediated resistance to tick bites, a phenomenon known as tick-immunity. An I. scapularis salivary gland cDNA expression library was therefore probed with sera from tick-immune guinea pigs to identify antigens that elicit humoral responses in the host. Sera from sensitized guinea pigs strongly recognized 3 of 4,500 library clones in an initial screening. The open reading frames of all 3 clones encoded a putative 16.4-kD acidic protein, designated Salp16, with an N-terminal signal sequence and signal peptidase cleavage sites specific for secretory proteins. The salp16 mRNA and Salp16 protein were detected in the salivary glands of engorged, but not unfed, nymphal and adult ticks, and Salp16 was also found in the saliva of engorged ticks. Immunization with recombinant Salp16 induced high antibody titers in guinea pigs, but did not elicit tick-immunity. Salp16 is the first feeding inducible gene that has been cloned from L. scapularis. Molecular characterization of I. scapularis salivary antigens that are induced upon tick feeding should help to facilitate our understanding of tick-host interactions.

Leptospiral outer membrane proteins OmpL1 and LipL41 exhibit synergistic immunoprotection.

New vaccine strategies are needed for prevention of leptospirosis, a widespread human and veterinary disease caused by invasive spirochetes belonging to the genus Leptospira. We have examined the immunoprotective capacity of the leptospiral porin OmpL1 and the leptospiral outer membrane lipoprotein LipL41 in the Golden Syrian hamster model of leptospirosis. Specialized expression plasmids were developed to facilitate expression of leptospiral proteins in Escherichia coli as the membrane-associated proteins OmpL1-M and LipL41-M. Although OmpL1-M expression is highly toxic in E. coli, this was accomplished by using plasmid pMMB66-OmpL1, which has undetectable background expression without induction. LipL41-M expression and processing were enhanced by altering its lipoprotein signal peptidase cleavage site to mimic that of the murein lipoprotein. Active immunization of hamsters with E. coli membrane fractions containing a combination of OmpL1-M and LipL41-M was found to provide significant protection against homologous challenge with Leptospira kirschneri serovar grippotyphosa. At 28 days after intraperitoneal inoculation, survival in animals vaccinated with both proteins was 71% (95% confidence interval [CI], 53 to 89%), compared with only 25% (95% CI, 8 to 42%) in the control group (P < 0.001). On the basis of serological, histological, and microbiological assays, no evidence of infection was found in the vaccinated survivors. The protective effects of immunization with OmpL1-M and LipL41-M were synergistic, since significant levels of protection were not observed in animals immunized with either OmpL1-M or LipL41-M alone. In contrast to immunization with the membrane-associated forms of leptospiral proteins, hamsters immunized with His(6)-OmpL1 and His(6)-LipL41 fusion proteins, either alone or in combination, were not protected. These data indicate that the manner in which OmpL1 and LipL41 associates with membranes is an important determinant of immunoprotection.

Secretion of xylanase A2 in Streptomyces lividans: dependence on signal peptides length, number and composition

The signal peptide (sp) in Streptomyces lividans xylanase A2 (XlnA2) was replaced by sps containing, in frame in their sequences, one, two, three or four initiation codons, each preceded by a Shine-Dalgarno (SD) sequence. Precursors of the corresponding proteins should thus have sps of, respectively, 27, 46, 82 and 91 amino acids (aa) long. By radiolabelling of S. lividans harboring the different constructs inserted in a multicopy plasmid and by immunoprecipitation with anti-xylanase antibodies followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation, precursors of the expected sizes were obtained in each clone. This indicates that ribosomes can synthesize different XlnA2 precursors from initiation codons inserted in the sp sequence, independently of their number. The amount of these synthesized precursors was also shown to be inversely proportional to their length when comparing the specific activity of labelling versus sp length. In clones producing more than one precursor, a smear appeared on the autoradiograms, suggesting some degree of precursor degradation. As determined by pulse-chase experiments, the rate of disappearance was almost the same for precursors of different lengths, but this might be the result of both true processing and proteolytic degradation. Furthermore, S. lividans rapidly degraded XlnA2 either when deprived of its sp or in the absence of the signal peptidase cleavage site.

A Novel Human Apolipoprotein (apoM)

A novel human apolipoprotein designated apolipoprotein M (apoM) is described. The unique N-terminal amino acid sequence of apoM was found in an approximately 26-kDa protein present in a protein extract of triglyceride-rich lipoproteins (TGRLP). The isolated apoM cDNA (734 base pairs) encoded a 188-amino acid residue-long protein, distantly related to the lipocalin family. The mRNA of apoM was detected in the liver and kidney. Western blotting demonstrated apoM to be present in high density lipoprotein (HDL) and to a lesser extent in TGRLP and low density lipoproteins (LDL). The first 20 amino acid residues of apoM constituted a hydrophobic segment with characteristic features of a signal peptide. This was retained in the mature protein because of the lack of a signal peptidase cleavage site. In vitro translation in the presence of microsomes demonstrated translocation of apoM over the membrane and glycosylation but no signal peptide cleavage. The in vitro translated product remained associated with the microsomes after treatment with carbonate at pH 11, demonstrating that apoM is an integral protein. This finding suggests that apoM is linked to the single phospholipid layer of lipoproteins with a hydrophobic signal anchor. In conclusion, a novel human apolipoprotein, the function of which remains to be determined, is described.

Secretion of xylanase A2 in Streptomyces lividans: dependence on signal peptides length, number and composition.

The signal peptide (sp) in Streptomyces lividans xylanase A2 (XlnA2) was replaced by sps containing, in frame in their sequences, one, two, three or four initiation codons, each preceded by a Shine-Dalgarno (SD) sequence. Precursors of the corresponding proteins should thus have sps of, respectively, 27, 46, 82 and 91 amino acids (aa) long. By radiolabelling of S. lividans harboring the different constructs inserted in a multicopy plasmid and by immunoprecipitation with anti-xylanase antibodies followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation, precursors of the expected sizes were obtained in each clone. This indicates that ribosomes can synthesize different XlnA2 precursors from initiation codons inserted in the sp sequence, independently of their number. The amount of these synthesized precursors was also shown to be inversely proportional to their length when comparing the specific activity of labelling versus sp length. In clones producing more than one precursor, a smear appeared on the autoradiograms, suggesting some degree of precursor degradation. As determined by pulse-chase experiments, the rate of disappearance was almost the same for precursors of different lengths, but this might be the result of both true processing and proteolytic degradation. Furthermore, S. lividans rapidly degraded XlnA2 either when deprived of its sp or in the absence of the signal peptidase cleavage site.

The disulfide-bonded loop of chromogranin B mediates membrane binding and directs sorting from the trans-Golgi network to secretory granules.

The disulfide-bonded loop of chromogranin B (CgB), a regulated secretory protein with widespread distribution in neuroendocrine cells, is known to be essential for the sorting of CgB from the trans-Golgi network (TGN) to immature secretory granules. Here we show that this loop, when fused to the constitutively secreted protein alpha1-antitrypsin (AT), is sufficient to direct the fusion protein to secretory granules. Importantly, the sorting efficiency of the AT reporter protein bearing two loops (E2/3-AT-E2/3) is much higher compared with that of AT with a single disulfide-bonded loop. In contrast to endogenous CgB, E2/3-AT-E2/3 does not undergo Ca2+/pH-dependent aggregation in the TGN. Furthermore, the disulfide-bonded loop of CgB mediates membrane binding in the TGN and does so with 5-fold higher efficiency if two loops are present on the reporter protein. The latter finding supports the concept that under physiological conditions, aggregates of CgB are the sorted units of cargo which have multiple loops on their surface leading to high membrane binding and sorting efficiency of CgB in the TGN.

Topology of Euglena Chloroplast Protein Precursors within Endoplasmic Reticulum to Golgi to Chloroplast Transport Vesicles

Euglena chloroplast protein precursors are transported as integral membrane proteins from the endoplasmic reticulum (ER) to the Golgi apparatus prior to chloroplast localization. All Euglena chloroplast protein precursors have functionally similar bipartite presequences composed of an N-terminal signal peptide domain and a stromal targeting domain containing a hydrophobic region approximately 60 amino acids from the predicted signal peptidase cleavage site. Asparagine-linked glycosylation reporters and presequence deletion constructs of the precursor to the Euglena light-harvesting chlorophyll a/b-binding protein of photosystem II (pLHCPII) were used to identify presequence regions translocated into the ER lumen and stop transfer membrane anchor domains. An asparagine-linked glycosylation site present at amino acid 148 of pLHCPII near the N terminus of mature LHCPII was not glycosylated in vitro by canine microsomes while an asparagine-linked glycosylation site inserted at amino acid 40 was. The asparagine at amino acid 148 was glycosylated upon deletion of amino acids 46-146, which contain the stromal targeting domain, indicating that the hydrophobic region within this domain functions as a stop transfer membrane anchor sequence. Protease protection assays indicated that for all constructs, mature LHCPII was not translocated across the microsomal membrane. Taken together with the structural similarity of all Euglena presequences, these results demonstrate that chloroplast precursors are anchored within ER and Golgi transport vesicles by the stromal targeting domain hydrophobic region oriented with the presequence N terminus formed by signal peptidase cleavage in the vesicle lumen and the mature protein in the cytoplasm.

Influenza C virus CM2 integral membrane glycoprotein is produced from a polypeptide precursor by cleavage of an internal signal sequence.

The influenza C virus CM2 protein is a small glycosylated integral membrane protein (115 residues) that spans the membrane once and contains a cleavable signal sequence at its N terminus. The coding region for CM2 (CM2 ORF) is located at the C terminus of the 342-amino acid (aa) ORF of a colinear mRNA transcript derived from influenza C virus RNA segment 6. Splicing of the colinear transcript introduces a translational stop codon into the ORF and the spliced mRNA encodes the viral matrix protein (CM1) (242 aa). The mechanism of CM2 translation was investigated by using in vitro and in vivo translation of RNA transcripts. It was found that the colinear mRNA derived from influenza C virus RNA segment 6 serves as the mRNA for CM2. Furthermore, CM2 translation does not depend on any of the three in-frame methionine residues located at the beginning of CM2 ORF. Rather, CM2 is a proteolytic cleavage product of the p42 protein product encoded by the colinear mRNA: a cleavage event that involves the recognition and cleavage of an internal signal peptide presumably by signal peptidase resident in the endoplasmic reticulum. Alteration of the predicted signal peptidase cleavage site by mutagenesis blocked generation of CM2. The other polypeptide species resulting from the cleavage of p42, designated p31, contains the CM1 coding region and an additional C-terminal 17 aa (formerly the CM2 signal peptide). Protein p31, in comparison to CM1, displays characteristics of an integral membrane protein.

Extracellular and cytoplasmic Cu/Zn superoxide dismutases from Haemonchus contortus.

Full-length cDNAs encoding cytosolic (SODc) and putative extracellular (SODe) Cu/Zn superoxide dismutases (SODs) from the ovine gastrointestinal parasitic nematode Haemonchus contortus have been isolated and characterized. The predicted sequences of the H. contortus SODs showed strong homology to other helminth SODs, the highest level of sequence similarity was with those of the free-living nematode Caenorhabditis elegans++. The predicted amino acid sequence of the putative extracellular form contained an N-terminal extension with the characteristics of a signal sequence including a potential signal peptidase cleavage site. Transcripts of both classes of Cu/Zn SOD were detected in all life-cycle stages examined. The cytosolic SOD mRNA was approximately 6-fold more abundant than that of the extracellular enzyme in adult parasites. Immunoblotting with antisera raised to in vitro-expressed parasite SODs revealed the presence of 2 proteins in extracts of adult H. contortus, with molecular masses of approximately 19.8 and 18 kDa. An additional protein of approximately 16.8 kDa was detected in adult ES material. Immunofluorescent staining showed Cu/Zn SOD was localized in the body wall musculature and the pharynx in adult worms and in the uterine tract of adult females. The immunogenic properties of recombinant H. contortus Cu/Zn SODs was assessed in a challenge infection experiment in lambs.

Characterization of leptospiral outer membrane lipoprotein LipL36: downregulation associated with late-log-phase growth and mammalian infection.

We report the cloning of the gene encoding a 36-kDa leptospiral outer membrane lipoprotein, designated LipL36. We obtained the N-terminal amino acid sequence of a staphylococcal V8 proteolytic-digest fragment in order to design an oligonucleotide probe. A Lambda-Zap II library containing EcoRI fragments of Leptospira kirschneri DNA was screened, and a 2.3-kb DNA fragment which contained the entire structural lipL36 gene was identified. Several lines of evidence indicate that LipL36 is lipid modified in a manner similar to that of LipL41, a leptospiral outer membrane lipoprotein we described in a previous study (E. S. Shang, T. A. Summers, and D. A. Haake, Infect. Immun. 64:2322-2330, 1996). The deduced amino acid sequence of LipL36 would constitute a 364-amino-acid polypeptide with a 20-amino-acid signal peptide, followed by an L-X-Y-C lipoprotein signal peptidase cleavage site. LipL36 is solubilized by Triton X-114 extraction of L. kirschneri; phase separation results in partitioning of LipL36 exclusively into the hydrophobic, detergent phase. LipL36 is intrinsically labeled during incubation of L. kirschneri in media containing [3H]palmitate. Processing of LipL36 is inhibited by globomycin, a selective inhibitor of lipoprotein signal peptidase. After processing, LipL36 is exported to the outer membrane along with LipL41 and lipopolysaccharide. Unlike LipL41, there appears to be differential expression of LipL36. In early-log-phase cultures, LipL36 is one of the most abundant L. kirschneri proteins. However, LipL36 levels drop considerably beginning in mid-log phase. LipL36 expression in vivo was evaluated by examining the humoral immune response to leptospiral antigens in the hamster model of leptospirosis. Hamsters surviving challenge with culture-adapted virulent L. kirschneri generate a strong antibody response to LipL36. In contrast, sera from hamsters surviving challenge with host-adapted L. kirschneri do not recognize LipL36. These findings suggest that LipL36 expression is downregulated during mammalian infection, providing a marker for studying the mechanisms by which pathogenic Leptospira species adapt to the host environment.

Signal peptidase cleavage at the flavivirus C-prM junction: dependence on the viral NS2B-3 protease for efficient processing requires determinants in C, the signal peptide, and prM.

Signal peptidase cleavage at the C-prM junction in the flavivirus structural polyprotein is inefficient in the absence of the cytoplasmic viral protease, which catalyzes cleavage at the COOH terminus of the C protein. The signal peptidase cleavage occurs efficiently in circumstances where the C protein is deleted or if the viral protease complex is present. In this study, we used cDNA of Murray Valley encephalitis virus (MVE) to examine features of the structural polyprotein which allow this regulation of a luminal cleavage by a cytoplasmic protease. We found that the inefficiency of signal peptidase cleavage in the absence of the viral protease is not attributable solely to features of the C protein. Inhibition of cleavage still occurred when charged residues in C were mutated to uncharged residues or when an unrelated protein sequence (that of ubiquitin) was substituted for C. Also, fusion of the C protein did not inhibit processing of an alternative adjacent signal sequence. The cleavage region of the flavivirus prM translocation signal is unusually hydrophobic, and we established that altering this characteristic by making three point mutations near the signal peptidase cleavage site in MVE prM dramatically increased the extent of cleavage without requiring removal of the C protein. In addition, we demonstrated that luminal sequences downstream from the signal peptidase cleavage site contributed to the inefficiency of cleavage.

Isolation, sequencing and expression of the gene encoding a major protein from the backteriophage associated with Bartonella henselae

The gene encoding a 31-kDa major protein (Pap31) associated with the bacteriophage harbored in Bartonella henselae was cloned and sequenced. Analysis of the resulting sequence revealed an open reading frame of 837 nucleotides coding for a protein of 279 amino acids. pap31 was then subcloned downstream of the lacZ promoter in pUC19. pap31 was amplified by polymerase chain reaction, and the linear amplicon was used as template for in-vitro transcription and translation. A protein with an apparent molecular mass of approximately 31 kDa was synthesized from this reaction. Upon analysis of the deduced aa sequence, a potential signal sequence and a consensus signal peptidase cleavage site were identified, indicative that Pap31 is modified posttranslationally, and the mature protein may be targeted to the host membrane.

Molecular cloning and N-terminal analysis of bovine cystatin C: Identification of a full-length N-terminal region .

The N-terminal region of human cystatin C has been shown to be of crucial importance for the interaction of the inhibitor with cysteine proteinases. However, several studies have been unable to identify the corresponding region in bovine cystatin C, indicating that the binding of proteinases to the bovine inhibitor may not be dependent on this region. With the aim to resolve this apparent discrepancy and to elucidate the relation of bovine cystatin C to other cystatins, we have isolated a cDNA clone encoding bovine precystatin C. The sequence of this cDNA was similar to that of the human inhibitor and showed a putative signal peptidase cleavage site consistent with the N-terminal regions of the bovine and human inhibitors being of comparable size. This suggestion was verified by determination of the relative molecular mass of the mature bovine inhibitor isolated from cerebrospinal fluid under conditions minimising proteolysis. The N-terminal of the purified inhibitor was blocked, but the sequence of the N-terminal peptide produced by digestion with endopeptidase LysC could be unequivocally determined by tandem mass spectroscopy. Together, these results show that bovine cystatin C has 118 residues, in contrast with 110–112 residues reported previously, and has an N-terminal region analogous to that of human cystatin C. This region presumably is of similar importance for tight binding of target proteinases as in the human inhibitor.

Mouse Extracellular Superoxide Dismutase: Primary Structure, Tissue-specific Gene Expression, Chromosomal Localization, and Lung In Situ Hybridization

Extracellular superoxide dismutase (EC-SOD) is the major extracellular antioxidant enzyme. We have determined the primary structure of mouse EC-SOD by characterization of complementary DNA (cDNA) clones and by amino-acid sequence analysis of purified protein. cDNA sequence analysis indicates that mouse EC-SOD is synthesized as a 251-amino-acid precursor protein with a predicted molecular weight of 27,400 D. Amino-terminal micro sequence analysis of purified mature mouse lung EC-SOD demonstrated the sequence to begin with SSFDLADRLDPV-. These results indicate that EC-SOD as initially synthesized contains a 24-amino-acid precursor peptide, and that the mature protein is 227 amino acids in length. Computer algorithms that predict the most likely site of cotranslational signal peptidase cleavage suggest that processing will occur between amino acids 18 and 19 or 20 and 21, which implies that EC-SOD may be initially synthesized as a pre-pro-protein. Like human EC-SOD, mature mouse EC-SOD is glycosylated. The full-length mouse EC-SOD cDNA is 1,834 base pairs long and is 82% (79% for protein) identical to rat EC-SOD, but only 60% (60% for protein) identical to human EC-SOD. The mouse EC-SOD gene locus (Sod3) was mapped by interspecific backcross haplotype analysis as being 0.9 +/- 0.9 centimorgans distal to the Qdpr locus on mouse Chromosome 5, a position suggesting that the human homologue of EC-SOD will map close to the human QDPR locus (4p15.3). Of nine tissues examined by Northern blot analysis, those of the kidney and lung are by far the major tissues that express EC-SOD messenger RNA. Using in situ hybridization in the mouse lung, we demonstrate EC-SOD gene expression to be highly localized to alveolar Type II epithelial cells. These data suggest that alveolar Type II cells play a central role in mediating EC-SOD antioxidant function in the lung.

Complementation Studies with Co-expressed Fragments of the Human Red Cell Anion Transporter (Band 3; AE1): THE ROLE OF SOME EXOFACIAL LOOPS IN ANION TRANSPORT

We constructed cDNA clones encoding fragments of band 3 in which the membrane domain was truncated from either the N or the C terminus within each of the first four exofacial loops. The truncations containing the C terminus of the protein were fused with the cleavable N-terminal signal sequence of glycophorin A to facilitate the correct orientation of the most N-terminal band 3 membrane span. Cleavage of the glycophorin A signal sequence was observed, except when the truncation was in the first exofacial loop where the signal peptidase cleavage site was probably too close to the membrane. The anion transport activity of co-expressed complementary pairs of truncations which together contained the entire band 3 membrane domain was examined. The pairs of fragments divided in the third and fourth exofacial loops yielded transport activity, but the pair separated within the second exofacial loop was not active. We conclude that the integrity of the second exofacial loop, but not the third and fourth exofacial loops, is necessary for transport activity. The unusually stable association between the fragments divided in the second exofacial loop suggests that interactions may occur between polar surfaces on amphiphilic portions of the third and fifth transmembrane spans.