Utilization of Escherichia coli Outer-Membrane Endoprotease OmpT Variants as Processing Enzymes for Production of Peptides from Designer Fusion Proteins

Escherichia coli outer-membrane endoprotease OmpT has suitable properties for processing fusion proteins to produce peptides and proteins. However, utilization of this protease for such production has been restricted due to its generally low cleavage efficiency at Arg (or Lys)-Xaa, where Xaa is a nonbasic N-terminal amino acid of a target polypeptide. The objective of this study was to generate a specific and efficient OmpT protease and to utilize it as a processing enzyme for producing various peptides and proteins by converting its substrate specificity. Since OmpT Asp⁹⁷ is proposed to interact with the P1′ amino acid of its substrates, OmpT variants with variations at Asp⁹⁷ were constructed by replacing this amino acid with 19 natural amino acids to alter the cleavage specificity at Arg (P1)-Xaa (P1′). The variant OmpT that had a methionine at this position, but not the wild-type OmpT, efficiently cleaved a fusion protein containing the amino acid sequence -Arg-Arg-Arg-Ala-Arg↓motilin, in which motilin is a model peptide with a phenylalanine at the N terminus. The OmpT variants with leucine and histidine at position 97 were useful in releasing human adrenocorticotropic hormone (1-24) (serine at the N terminus) and human calcitonin precursor (cysteine at the N terminus), respectively, from fusion proteins. Motilin was produced by this method and was purified up to 99.0% by two chromatographic steps; the yield was 160 mg/liter of culture. Our novel method in which the OmpT variants are used could be employed for production of various peptides and proteins.     

Substrate specificity of the Escherichia coli outer membrane protease OmpT

OmpT is a surface protease of gram-negative bacteria that has been shown to cleave antimicrobial peptides, activate human plasminogen, and degrade some recombinant heterologous proteins. We have analyzed the substrate specificity of OmpT by two complementary substrate filamentous phage display methods: (i) in situ cleavage of phage that display protease-susceptible peptides by Escherichia coli expressing OmpT and (ii) in vitro cleavage of phage-displayed peptides using purified enzyme. Consistent with previous reports, OmpT was found to exhibit a virtual requirement for Arg in the P1 position and a slightly less stringent preference for this residue in the P1' position (P1 and P1' are the residues immediately prior to and following the scissile bond). Lys, Gly, and Val were also found in the P1' position. The most common residues in the P2' position were Val or Ala, and the P3 and P4 positions exhibited a preference for Trp or Arg. Synthetic peptides based upon sequences selected by bacteriophage display were cleaved very efficiently, with kcat/Km values up to 7.3 x 10(6) M(-1) s(-1). In contrast, a peptide corresponding to the cleavage site of human plasminogen was hydrolyzed with a kcat/Km almost 10(6)-fold lower. Overall, the results presented in this work indicate that in addition to the P1 and P1' positions, additional amino acids within a six-residue window (between P4 and P2') contribute to the binding of substrate polypeptides to the OmpT binding site.     

Substrate specificity at the P1' site of Escherichia coli OmpT under denaturing conditions

Though OmpT has been reported to mainly cleave the peptide bond between consecutive basic amino acids, we identified more precise substrate specificity by using a series of modified substrates, termed PRX fusion proteins, consisting of 184 residues. The cleavage site of the substrate PRR was Arg140-Arg141 and the modified substrates PRX substituted all 19 natural amino acids at the P1' site instead of Arg141. OmpT under denaturing conditions (in the presence of 4 M urea) cleaved not only between two consecutive basic amino acids but also at the carboxyl side of Arg140 except for the Arg140-Asp141, -Glu141, and -Pro141 pairs. In addition to Arg140 at the P1 site, similar results were obtained when Lys140 was substituted into the P1 site. In the absence of urea, an aspartic acid residue at the P1' site was unfavorable for OmpT cleavage of synthetic decapeptides, the enzyme showed a preference for a dibasic site.     

Substrate specificity of the Escherichia coli outer membrane protease OmpP

Escherichia coli OmpP is an F episome-encoded outer membrane protease that exhibits 71% amino acid sequence identity with OmpT. These two enzymes cleave substrate polypeptides primarily between pairs of basic amino acids. We found that, like OmpT, purified OmpP is active only in the presence of lipopolysaccharide. With optimal peptide substrates, OmpP exhibits high catalytic efficiency (k(cat)/K(m) = 3.0 x 10(6) M(-1)s(-1)). Analysis of the extended amino acid specificity of OmpP by substrate phage revealed that both Arg and Lys are strongly preferred at the P1 and P1' sites of the enzyme. In addition, Thr, Arg, or Ala is preferred at P2; Leu, Ala, or Glu is preferred at P4; and Arg is preferred at P3'. Notable differences in OmpP and OmpT specificities include the greater ability of OmpP to accept Lys at the P1 or P1', site as well as the prominence of Ser at P3 in OmpP substrates. Likewise, the OmpP P1 site could better accommodate Ser; as a result, OmpP was able to cleave a peptide substrate between Ser-Arg about 120 times more efficiently than was OmpT. Interestingly, OmpP and OmpT cleave peptides with three consecutive Arg residues at different sites, a difference in specificity that might be important in the inactivation of cationic antimicrobial peptides. Accordingly, we show that the presence of an F' episome results in increased resistance to the antimicrobial peptide protamine both in ompT mutants and in wild-type E. coli cells.     

Substrate specificity of the integral membrane protease OmpT determined by spatially addressed peptide libraries

Escherichia coli outer membrane protease T (OmpT) is an endopeptidase that specifically cleaves between two consecutive basic residues. In this study we have investigated the substrate specificity of OmpT using spatially addressed SPOT peptide libraries. The peptide acetyl-Dap(dnp)-Ala-Arg/Arg-Ala-Lys(Abz)-Gly was synthesized directly onto cellulose membrane. The peptide contained the aminobenzoyl (Abz) fluorophore, which was internally quenched by the dinitrophenyl (dnp) moiety. Treatment of the SPOT membrane with the small, water-soluble protease trypsin resulted in highly fluorescent peptide SPOTs. However, no peptide cleavage was observed after incubation with detergent-solubilized OmpT, a macromolecular complex with an estimated molecular mass of 180 kDa. This problem could be solved by the introduction of a long, polar polyoxyethylene glycol linker between the membrane support and the peptide. Peptide libraries for the P(2), P(1), P(1)', and P(2)' positions in the substrate were screened with OmpT, and peptides of positive SPOTs were resynthesized and subjected to kinetic measurements in solution. The best substrate Abz-Ala-Lys-Lys-Ala-Dap(dnp)-Gly had a turnover number k(cat) of 40 s(-)(1), which is 12-fold higher than the starting substrate. Peptides containing an acidic residue at P(2) or P(2)' were not substrates for OmpT, suggesting that long-range electrostatic interactions are important for the formation of the enzyme-substrate complex. OmpT was highly selective toward L-amino acids at P(1) but was less so at P(1)' where a peptide with D-Arg at P(1)' was a competitive inhibitor (K(i) of 19 microM). An affinity chromatography resin based on these findings was developed, which allowed for the one-step purification of OmpT from a bacterial lysate. The implications of the determined consensus substrate sequence (Arg/Lys)/(Arg/Lys)-Ala for the proposed biological function of OmpT in defense against antimicrobial peptides are discussed.     

Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.     

Effect of the N1 residue on the stability of the alpha-helix for all 20 amino acids

N1 is the first residue in an alpha-helix. We have measured the contribution of all 20 amino acids to the stability of a small helical peptide CH(3)CO-XAAAAQAAAAQAAGY-NH(2) at the N1 position. By substituting every residue into the N1 position, we were able to investigate the stabilizing role of each amino acid in an isolated context. The helix content of each of the 20 peptides was measured by circular dichroism (CD) spectroscopy. The data were analyzed by our modified Lifson-Roig helix-coil theory, which includes the n1 parameter, to find free energies for placing a residue into the N1 position. The rank order for free energies is Asp(-), Ala > Glu(-) > Glu(0) > Trp, Leu, Ser > Asp(0), Thr, Gln, Met, Ile > Val, Pro > Lys(+), Arg, His(0) > Cys, Gly > Phe > Asn, Tyr, His(+). N1 preferences are clearly distinct from preferences for the preceding N-cap and alpha-helix interior. pK(a) values were measured for Asp, Glu, and His, and protonation-free energies were calculated for Asp and Glu. The dissociation of the Asp proton is less favorable than that of Glu, and this reflects its involvement in a stronger stabilizing interaction at the N terminus. Proline is not energetically favored at the alpha-helix N terminus despite having a high propensity for this position in crystal structures. The data presented are of value both in rationalizing mutations at N1 alpha-helix sites in proteins and in predicting the helix contents of peptides.     

Side-chain structures in the first turn of the alpha-helix

The first three residues at the N terminus of the alpha-helix are called N1, N2 and N3. We surveyed 2102 alpha-helix N termini in 298 high-resolution, non-homologous protein crystal structures for N1, N2 and N3 amino acid and side-chain rotamer propensities and hydrogen-bonding patterns. We find strong structural preferences that are unique to these sites. The rotamer distributions as a function of amino acid identity and position in the helix are often explained in terms of hydrogen-bonding interactions to the free N1, N2 and N3 backbone NH groups. Notably, the "good N2" amino acid residues Gln, Glu, Asp, Asn, Ser, Thr and His preferentially form i, i or i,i+1 hydrogen bonds to the backbone, though this is hindered by good N-caps (Asp, Asn, Ser, Thr and Cys) that compete for these hydrogen bond donors. We find a number of specific side-chain to side-chain interactions between N1 and N2 or between the N-cap and N2 or N3, such as Arg(N-cap) to Asp(N2). The strong energetic and structural preferences found for N1, N2 and N3, which differ greatly from positions within helix interiors, suggest that these sites should be treated explicitly in any consideration of helical structure in peptides or proteins.     

3C-like protease of rabbit hemorrhagic disease virus: identification of cleavage sites in the ORF1 polyprotein and analysis of cleavage specificity.

Rabbit hemorrhagic disease virus, a positive-stranded RNA virus of the family Caliciviridae, encodes a trypsin-like cysteine protease as part of a large polyprotein. Upon expression in Escherichia coli, the protease releases itself from larger precursors by proteolytic cleavages at its N and C termini. Both cleavage sites were determined by N-terminal sequence analysis of the cleavage products. Cleavage at the N terminus of the protease occurred with high efficiency at an EG dipeptide at positions 1108 and 1109. Cleavage at the C terminus of the protease occurred with low efficiency at an ET dipeptide at positions 1251 and 1252. To study the cleavage specificity of the protease, amino acid substitutions were introduced at the P2, P1, and P1' positions at the cleavage site at the N-terminal boundary of the protease. This analysis showed that the amino acid at the P1 position is the most important determinant for substrate recognition. Only glutamic acid, glutamine, and aspartic acid were tolerated at this position. At the P1' position, glycine, serine, and alanine were the preferred substrates of the protease, but a number of amino acids with larger side chains were also tolerated. Substitutions at the P2 position had only little effect on the cleavage efficiency. Cell-free expression of the C-terminal half of the ORF1 polyprotein showed that the protease catalyzes cleavage at the junction of the RNA polymerase and the capsid protein. An EG dipeptide at positions 1767 and 1768 was identified as the putative cleavage site. Our data show that rabbit hemorrhagic disease virus encodes a trypsin-like cysteine protease that is similar to 3C proteases with regard to function and specificity but is more similar to 2A proteases with regard to size.     

High affinity small protein inhibitors of human chymotrypsin C (CTRC) selected by phage display reveal unusual preference for P4' acidic residues

Human chymotrypsin C (CTRC) is a pancreatic protease that participates in the regulation of intestinal digestive enzyme activity. Other chymotrypsins and elastases are inactive on the regulatory sites cleaved by CTRC, suggesting that CTRC recognizes unique sequence patterns. To characterize the molecular determinants underlying CTRC specificity, we selected high affinity substrate-like small protein inhibitors against CTRC from a phage library displaying variants of SGPI-2, a natural chymotrypsin inhibitor from Schistocerca gregaria. On the basis of the sequence pattern selected, we designed eight inhibitor variants in which amino acid residues in the reactive loop at P1 (Met or Leu), P2' (Leu or Asp), and P4' (Glu, Asp, or Ala) were varied. Binding experiments with CTRC revealed that (i) inhibitors with Leu at P1 bind 10-fold stronger than those with P1 Met; (ii) Asp at P2' (versus Leu) decreases affinity but increases selectivity, and (iii) Glu or Asp at P4' (versus Ala) increase affinity 10-fold. The highest affinity SGPI-2 variant (K(D) 20 pm) bound to CTRC 575-fold tighter than the parent molecule. The most selective inhibitor variant exhibited a K(D) of 110 pm and a selectivity ranging from 225- to 112,664-fold against other human chymotrypsins and elastases. Homology modeling and mutagenesis identified a cluster of basic amino acid residues (Lys(51), Arg(56), and Arg(80)) on the surface of human CTRC that interact with the P4' acidic residue of the inhibitor. The acidic preference of CTRC at P4' is unique among pancreatic proteases and might contribute to the high specificity of CTRC-mediated digestive enzyme regulation.     

Strategy for improvement of enteropeptidase efficiency in tag removal processes

Enteropeptidase (synonym: enterokinase, EC 3.4.21.9) is a heterodimeric serine protease of the intestinal brush border that activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the sequence (Asp)(4)-Lys. It has also great biotechnological interest because of the unique substrate specificity of the serine protease domain. The high degree of specificity exhibited by enteropeptidase makes it a suitable reagent for cleaving recombinant proteins to remove affinity or other tags. However often unwanted cleavages elsewhere in the protein occurred during cleavage of fusions when high amount of enzyme is required. In this study we have improved the efficiency of fusion proteins cleavage by enteropeptidase by substitution of the Lys residue by Arg in specific cleavage sequence (Asp)(4)-Lys. We have demonstrated that 3-6-fold lower amounts of the catalytic subunit of human and bovine enteropeptidase is required for 95% cleavage of Trx/TRAIL and Trx/FGF-2 fusions with (Asp)(4)-Arg cleavage sequence in comparison to native sequence (Asp)(4)-Lys. As a result, reduced amount of non-specifically cleaved peptide fragments were observed during cleavage of (Asp)(4)-Lys/Arg mutated fusions. These findings overcome limitations of enteropeptidase in tag removal processes during recombinant proteins purification and extend its commercial benefit in the biopharmaceutical industry.     

High-Level Production of Recombinant Human Parathyroid Hormone 1-34

Expression of the synthetic human parathyroid hormone 1-34 [hPTH(1-34)] gene by a gene fusion strategy was demonstrated. hPTH(1-34) was produced at the C terminus of the partner peptides involving amino acids 1 to 97, 1 to 117, or 1 to 139 of a modified Escherichia coli β-galactosidase by linker peptides containing oligohistidine of different lengths. The fusion proteins in the inclusion bodies were rendered soluble with urea and subjected to site-specific cleavage with the secretory type yeast Kex2 protease. Optimal expression and enzymatic processing were achieved in the fusion protein βG-117S4HPT, constructed from amino acids 1 to 117 of β-galactosidase and the linker of HHHHPGGSVKKR. The fusion protein accumulated more than 20% of the E. coli total protein. The hPTH(1-34) was purified up to 99.5% with a good yield of 0.5 g/liter of culture. The purified product was identified as intact hPTH(1-34) by amino acid analysis and N-terminal sequencing.     

Identification and quantification of Fc fusion peptibody degradations by limited proteolysis method

An Fc fusion protein expressed in Escherichia coli contains Met1 and Asp2 residues at the N terminus and an active peptide attached to the C terminus of the Fc region. Due to the unique amino acid sequence of Fc, many commonly used proteolysis methods have severe drawbacks for characterizing degradations of Met1 and Asp2 residues. A novel method has been developed to effectively characterize the degradations by employing a limited endoproteinase Glu-C digestion. The limited digestion generates a dimeric peptide of (Met1-Glu14)(2) due to specific cleavage at the residue Glu14 of the N terminus. This peptide together with its degraded products, including Met1 oxidation and Asp2 isomerization, can be identified and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The optimization of digestion procedure and linearity of quantification are also described. This approach was successfully used in a photostability study to assess the product stability of an Fc fusion peptibody.     

IL-1-converting enzyme requires aspartic acid residues for processing of the IL-1 beta precursor at two distinct sites and does not cleave 31-kDa IL-1 alpha

IL-1 converting enzyme (ICE) specifically cleaves the human IL-1 beta precursor at two sequence-related sites: Asp27-Gly28 (site 1) and Asp116-Ala117 (site 2). Cleavage at Asp116-Ala117 results in the generation of mature, biologically active IL-1 beta. ICE is unusual in that preferred cleavage at Asp-X bonds (where X is a small hydrophobic residue), has not been described for any other eukaryotic protease. To further examine the substrate specificity of ICE, proteins that contain Asp-X linkages including transferrin, actin, complement factor 9, the murine IL-1 beta precursor, and human and murine IL-1 alpha precursors, were assayed for cleavage by 500-fold purified ICE. The human and murine IL-1 beta precursors were the only proteins cleaved by ICE, demonstrating that ICE is an IL-1 beta convertase. Analysis of human IL-1 beta precursor mutants containing amino acid substitutions or deletions within each processing site demonstrated that omission or replacement of Asp at site 1 or site 2 prevented cleavage by ICE. To quantitatively assess the substrate requirements of ICE, a peptide-based cleavage assay was established using a 14-mer spanning site 2. Cleavage between Asp [P1] and Ala [P1']2 was demonstrated. Replacement of Asp with Ala, Glu, or Asn resulted in a greater than 100-fold reduction in cleavage activity. The rank order in position P1' was Gly greater than Ala much greater than Leu greater than Lys greater than Glu. Substitutions at P2'-P4' and P6' had relatively little effect on cleavage activity. These results show that ICE is a highly specific IL-1 beta convertase with absolute requirements for Asp in P1 and a small hydrophobic amino acid in P1'.     

Studies on Nα-acylated proteins: The N-terminal sequences of two muscle enolases

A standard procedure for the identification of the N-terminal amino acid in N alpha-acylated proteins has been developed. After exhaustive proteolysis, the amino acids with blocked alpha-amino groups are separated from positively charged, free amino acids by ion exchange chromatography and subjected to digestion with acylase I. Amino acid analysis before and after the acylase treatment identifies the blocked N-terminal amino acid. A survey of acylamino acid substrates showed that acylase will liberate all the common amino acids except Asp, Cys or Pro from their N-acetyl-and N-butyryl derivatives, and will also catalyze the hydrolysis of N-formyl-Met and N-myristyl-Val. Thus, the procedure cannot identify acylated Asp, Cys or Pro, nor, because of the ion exchange step, N alpha-acyl-derivatives of Arg, Lys or His. Whenever the protease treatment releases free acylamino acids, the remaining amino acids should be detected. When applied to several proteins, the procedure confirmed known N-terminal acylamino acids and identified acyl-Ser in enolases from chum and coho salmon muscle and in pyruvate kinase from rabbit muscle, and acyl-Thr in phosphofructokinase from rabbit muscle. The protease-acylase assay has been used to identify blocked peptides from CNBr- or protease-treated proteins. When such peptides were treated with 1 N HCl at 110 degrees for 10 min, sufficient yields of deacylated, mostly intact, peptide were obtained to permit direct automatic sequencing. The N-terminal sequences of rabbit muscle and coho salmon enolase were determined in this way and are compared to each other and to the sequence of yeast enolase.     

Molecular basis for the relative substrate specificity of human immunodeficiency virus type 1 and feline immunodeficiency virus proteases

We have used a random hexamer phage library to delineate similarities and differences between the substrate specificities of human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) proteases (PRs). Peptide sequences were identified that were specifically cleaved by each protease, as well as sequences cleaved equally well by both enzymes. Based on amino acid distinctions within the P3-P3' region of substrates that appeared to correlate with these cleavage specificities, we prepared a series of synthetic peptides within the framework of a peptide sequence cleaved with essentially the same efficiency by both HIV-1 and FIV PRs, Ac-KSGVF/VVNGLVK-NH(2) (arrow denotes cleavage site). We used the resultant peptide set to assess the influence of specific amino acid substitutions on the cleavage characteristics of the two proteases. The findings show that when Asn is substituted for Val at the P2 position, HIV-1 PR cleaves the substrate at a much greater rate than does FIV PR. Likewise, Glu or Gln substituted for Val at the P2' position also yields peptides specifically susceptible to HIV-1 PR. In contrast, when Ser is substituted for Val at P1', FIV PR cleaves the substrate at a much higher rate than does HIV-1 PR. In addition, Asn or Gln at the P1 position, in combination with an appropriate P3 amino acid, Arg, also strongly favors cleavage by FIV PR over HIV PR. Structural analysis identified several protease residues likely to dictate the observed specificity differences. Interestingly, HIV PR Asp30 (Ile-35 in FIV PR), which influences specificity at the S2 and S2' subsites, and HIV-1 PR Pro-81 and Val-82 (Ile-98 and Gln-99 in FIV PR), which influence specificity at the S1 and S1' subsites, are residues which are often involved in development of drug resistance in HIV-1 protease. The peptide substrate KSGVF/VVNGK, cleaved by both PRs, was used as a template for the design of a reduced amide inhibitor, Ac-GSGVF Psi(CH(2)NH)VVNGL-NH(2.) This compound inhibited both FIV and HIV-1 PRs with approximately equal efficiency. These findings establish a molecular basis for distinctions in substrate specificity between human and feline lentivirus PRs and offer a framework for development of efficient broad-based inhibitors.     

Display and release of the Plasmodium falciparum circumsporozoite protein using the autotransporter MisL of Salmonella enterica

The Salmonella enterica MisL (protein of membrane insertion and secretion) is an autotransporter with high homology to AIDA-I (adhesin involved in diffuse adherence) of enteropathogenic Escherichia coli. Considering that it has been reported that the MisL beta translocator domain is able to display heterologous passenger peptides to the bacterial surface, we developed a system to display proteins and release them to the external environment by means of proteolytic cleavage. Plasmids were constructed encoding 8 or 53 repeats of the NANP (Asp-Ala-Asp-Pro) tetrapeptide, which is the main B cell epitope of the Plasmodium falciparum circumsporozoitic protein (CSP), fused to the the MisL beta-domain and including the recognition cleavage sequence from the E. coli OmpT surface protease. E. coli XL-10Gold and BL21(DE3) (OmpT positive and negative, respectively) and Salmonella enterica serovar Typhimurium SL3261 (Aro A(-)) were transformed with the plasmids and, both expression and localization of the fusion proteins were assessed by Western blot, indirect immunofluorescence, and flow cytometry, using a monoclonal antibody against (NANP)(3). Higher expression of the (NANP)(8) and (NANP)(53) fusion proteins was demonstrated on the bacterial surface of the OmpT negative E. coli strains and the (NANP)(53) in the culture supernatant of E. coli XL-10Gold indicating a protease mediated cleavage. The flow cytometry analysis suggested 71 and 98% cleavage efficiency for the (NANP)(8) and (NANP)(53), respectively, in E. coli XL-10Gold. Similar results were obtained in S. enterica serovar Typhimurium SL3261, suggesting the involvement of other proteases related to OmpT. These results demonstrate that MisL may be used for the autodisplay and release of passenger proteins in attenuated Salmonella or E. coli strains, which may have several applications in vaccine design.     

Functional analysis of the putative fusion domain of the baculovirus envelope fusion protein F

Group II nucleopolyhedroviruses (NPVs), e.g., Spodoptera exigua MNPV, lack a GP64-like protein that is present in group I NPVs but have an unrelated envelope fusion protein named F. In contrast to GP64, the F protein has to be activated by a posttranslational cleavage mechanism to become fusogenic. In several vertebrate viral fusion proteins, the cleavage activation generates a new N terminus which forms the so-called fusion peptide. This fusion peptide inserts in the cellular membrane, thereby facilitating apposition of the viral and cellular membrane upon sequential conformational changes of the fusion protein. A similar peptide has been identified in NPV F proteins at the N terminus of the large membrane-anchored subunit F(1). The role of individual amino acids in this putative fusion peptide on viral infectivity and propagation was studied by mutagenesis. Mutant F proteins with single amino acid changes as well as an F protein with a deleted putative fusion peptide were introduced in gp64-null Autographa californica MNPV budded viruses (BVs). None of the mutations analyzed had an major effect on the processing and incorporation of F proteins in the envelope of BVs. Only two mutants, one with a substitution for a hydrophobic residue (F152R) and one with a deleted putative fusion peptide, were completely unable to rescue the gp64-null mutant. Several nonconservative substitutions for other hydrophobic residues and the conserved lysine residue had only an effect on viral infectivity. In contrast to what was expected from vertebrate virus fusion peptides, alanine substitutions for glycines did not show any effect.     

Rearrangement of terminal amino acid residues in peptides by protease-catalyzed intramolecular transpeptidation

Protease-catalyzed rearrangements of amino acid residues in peptides are observed during enzymatic digestion of proteins. When two enzyme-specific cleavage sites are within one or two residues of each other in the protein sequence, only one of the two sites usually is hydrolyzed by the protease, resulting in a peptide that contains an extra cleavage site near one of its termini. It is observed that in this type of peptide, the residues between the two cleavage sites often rearrange from one terminus of the peptide to the other terminus, catalyzed by the protease that created the peptide. It is proposed that the rearrangement is caused by protease-catalyzed intramolecular transpeptidation through a cyclic peptide intermediate. Several cases of this type of rearrangement were observed for different peptides generated by different proteases, indicating that this type of rearrangement is a general phenomenon occurring during enzymatic digestion of proteins.     

The amino acid sequence of wheat histone H2B(2). A core histone with a novel repetitive N-terminal extension

Two of the four electrophoretic histone H2B variants present in wheat embryos have been isolated. The complete primary structure of the H2B(2) variant has been deduced from sets of overlapping peptides generated by CNBr cleavage, Staphylococcus aureus V8 protease, endoproteinase Arg-C, the post-proline cleaving enzyme, chymotrypsin and cleavage in dilute acid. A minimum of 17 peptides were required to establish the sequence. This variant has a blocked N terminus and comprises a total of 149 amino acids. The C-terminal two-thirds of the protein are highly homologous to vertebrate H2B. In contrast, the N-terminal third is entirely different and contains an N-terminal extension of 23 residues in which the sequence Ala-Glu-Lys or variants are repeated several times. This region is also highly homologous to the H2B from Tetrahymena pyriformis. It shows in addition similarities to wheat H2A(1) and bovine H1.