适合免疫治疗的大鼠抗肾小球基底膜肾炎模型的建立

以线性表位肽P14（a3_127-148）作为抗原建立适合免疫治疗的抗肾小球基底膜（anti-glomerular basement membrane,GBM）病大鼠模型。采用大鼠后脚垫三点注射P14（a3_127-148）与弗氏佐剂乳化物的方法进行单次免疫,免疫前后每周采集24 h尿样和血样,所有大鼠在免疫后7 w处死。大鼠免疫后,肾炎模型组在各时间点的24 h尿蛋白、尿蛋白肌酐比值（albumin/urine creatinine ratio,ACR）、血肌酐及血尿素氮均明显高于对照组（P<0.05）;循环抗P14（a3_127-148）IgG抗体水平明显高于对照组(P<0.001);PAS染色可见节段性纤维素样坏死,富于细胞型新月体;免疫组化染色可见肾小球有明显的中性粒细胞和巨噬细胞浸润;免疫荧光检测可见IgG沿GBM呈强线性沉积;电镜观察到GBM的断裂和收缩;而对照组均未见改变。HE染色在所有大鼠中均未观察到肺部病变。使用P14（a3_127-148）线性肽免疫WKY大鼠成功建立了大鼠抗GBM病模型,有助于开发更为特异的免疫疗法。     

Identifying N‐terminal peptides by a combination of the edman procedures with a bromine isotope tag: Application to the silicateins

Silicateins are proteins found within spicules of siliceous sponges. They are analogs of proteinases cathepsins; they catalyze the transformation of silicic acid esters into biogenic silica (SiO₂·nH₂O), and are believed to take part in the processes of silicification in marine and freshwater sponges. Earlier studies by Kalyuzhnaya et al. revealed that the Baikal Sponge Lubomirskia baicalensis Pallas, 1773 (L. baicalensis) contains a gene 1988 bp long, which hosts four sequences that encode four mRNAs giving rise to silicateins α1, α2, α3 and α4 (SILα1, SILα2, SILα3, SILα4) whose predicted amino acid sequences are similar to those of the predicted sequences of marine sponge silicateins. However, the sequences of mature silicateins of L. baicalensis remained unknown, since their N‐terminal peptides were not identified. We found the sequences of these N‐terminal peptides using a combination of the Edman procedure, which involved reaction with phenylisothiocyanate, treatment with trifluoroacetic acid and trypsinolysis followed by treatment with 4‐bromine‐phenylisothiocyanate performed directly within polyacrylamide gel bands, and subsequent mass spectrometry. The N‐terminal peptides are YAESIDWR (SILα1), YVDSIDWR (SILα2 and α4), and YADSLDWR (SILα3). All mature silicateins of L. baicalensis had a length 217 amino acid residues.     

Cystine-knot peptides engineered with specificities for α(IIb)β(3) or α(IIb)β(3) and α(v)β(3) integrins are potent inhibitors of platelet aggregation

A truncated form of the Agouti‐related protein (AgRP), a member of the cystine‐knot family, has shown promise as a scaffold for engineering novel peptides with new molecular recognition properties. In this study, we replaced a constrained six amino acid loop in AgRP with a nine amino acid loop containing an Arg–Gly–Asp integrin recognition motif, and randomized the neighboring residues to create a library of ～20 million AgRP variants. We displayed the AgRP mutants as fusions on the surface of yeast and used high‐throughput fluorescence‐activated cell sorting (FACS) to isolate peptides that bound specifically to the platelet integrin α_IIbβ₃, a clinically important target for the prevention and treatment of thrombosis. These AgRP peptides had equilibrium dissociation (K_D) constants for α_IIbβ₃ integrin ranging from 60 to 90 nM, and did not bind to α_vβ₃, α_vβ₅, or α₅β₁ integrins. Using an alternate library screening strategy, we identified AgRP peptides that bound to both α_IIbβ₃ and α_vβ₃ integrins with K_D values ranging from 40 to 70 nM and 20 to 30 nM, respectively, and did not bind to α_vβ₅ or α₅β₁ integrins. Unique consensus sequences were identified within both series of AgRP peptides suggesting alternative molecular recognition events that dictate different integrin binding specificities. In addition, the engineered AgRP peptides prevented platelet aggregation as well as or slightly better than the FDA‐approved cyclic peptide eptifibatide. Collectively, these data demonstrate that cystine‐knot peptides can be generated with high affinity and specificity to closely‐related integrins, and provide insights into molecular interactions between small, structured peptide ligands and their receptors. Copyright © 2010 John Wiley & Sons, Ltd.     

T cell epitope mimicry in antiglomerular basement membrane disease

Antiglomerular basement membrane (GBM) disease or Goodpasture's syndrome is among the earliest recognized human autoimmune diseases. Although collagen 4alpha3 NC1 (Col4alpha3NC1) has been identified as the responsible autoantigen, it remains unknown how autoimmunity to this autoantigen is provoked. We have demonstrated in our rat model that a single nephritogenic T cell epitope pCol28-40 of Col4alpha3NC1 induces glomerulonephritis. We hypothesized that microbial peptides that mimic this T cell epitope could induce the disease. Based on the critical residue motif (xxtTxNPsxx) of pCol28-40, seven peptides derived from human infection-related microbes were chosen through GenBank search and synthesized. All peptides showed cross-reactivity with pCol28-40-specific T cells at various levels. Only four peptides induced transient proteinuria and minor glomerular injury. However, the other three peptides induced severe proteinuria and modest to severe glomerulonephritis in 16-25% of the immunized rats. Unexpectedly, the most nephritogenic peptide, pCB, derived from Clostridium botulinum, also induced modest (25%) to severe (25%) pulmonary hemorrhage, another important feature of anti-GBM disease; this was not correlated with the severity of glomerulonephritis. This finding suggests that subtle variations in T cell epitope specificity may lead to different clinical manifestations of anti-GBM disease. In summary, our study raises the possibility that a single T cell epitope mimicry by microbial Ag may be sufficient to induce the anti-GBM disease.     

Proteolytically stable peptides by incorporation of α-Tfm amino acids

A series of model peptides containing α-trifluoromethyl-substituted amino acids in five different positions relative to the predominant cleavage site of the serine protease α-chymotrypsin was synthesized by solution methods to investigate the influence of α-Tfm substitution on the proteolytic stability of peptides. Proteolysis studies demonstrated absolute stability of peptides substituted in the P₁ position and still considerable proteolytic stability for peptides substituted at the P₂ and P′₂ positions compared with the corresponding unsubstituted model peptide. Comparison with peptides containing the fluorine-free disubstituted amino acid α-aminoisobutyric acid allowed to separate electronic from steric effects. Furthermore, the absolute configuration of the α-Tfm-substituted amino acid was found to exert considerable effects on the proteolytic stability, especially in P′₁ substituted peptides. Investigations of this phenomenon using empirical force field calculations revealed that in the (S,R,S)-diasteromer the steric constraints exhibited by the α-Tfm group can be outweighed by an advantageous interaction of the fluorine atoms with the serine side chain of the enzyme. In contrast, a favourable interaction between substrate and enzyme is impossible for the (S,S,S)-diastereomer. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

New insights into short peptides derived from the collagen NC1 α1, α2, and α3 (IV) domains: An experimental and MD simulations study

Proteolytically cleavage of the collagen NC1 α1 to α3 (IV) domains leads to antiangiogenic proteins called Arresten, Canstatin, and Tumstatin, respectively. The research identified that the two overlapping peptides derived from Tumstatin are more effective than other fragments and amino acids L78, V82, and D84 are essential for their activity. In the present study, the efficacy of a nine amino acid peptide derived from Tumstatin (Tum), containing amino acids L78, V82, and D84 was compared to the corresponding sequence in Arresten (Ars) and Canstatin (Can) in vitro and in vivo. Moreover, CD spectroscopy, MD, and docking simulations were performed to evaluate the structure and the interaction of peptides to integrin αvβ3, respectively. Results demonstrated that peptides inhibit viability, migration, and tube formation in vitro, as well as the growth of tumor in vivo and Canstatin-derived peptide was more potent than others. CD measurement and DSSP calculation revealed that Can had more coil conformation. According to MD simulations, Can had more fluctuation, less intramolecular interactions, and less structural compactness compared to Tum and Ars. It can be assumed that amino acid variations lead to a more flexible and loose structure compared to the other peptides. The Canstatin-derived peptide interacts with the integrin αvβ3 extremely close to RGD binding site by the most negative binding energy and more interactions. In conclusion, we for the first time identified an active peptide derived from Canstatin and showed that the sequence affected structure and thereby interaction of peptide to its receptor.     

Engineered cystine knot peptides that bind αvβ3, αvβ5, and α5β1 integrins with low‐nanomolar affinity

There is a critical need for compounds that target cell surface integrin receptors for applications in cancer therapy and diagnosis. We used directed evolution to engineer the Ecballium elaterium trypsin inhibitor (EETI‐II), a knottin peptide from the squash family of protease inhibitors, as a new class of integrin‐binding agents. We generated yeast‐displayed libraries of EETI‐II by substituting its 6‐amino acid trypsin binding loop with 11‐amino acid loops containing the Arg‐Gly‐Asp integrin binding motif and randomized flanking residues. These libraries were screened in a high‐throughput manner by fluorescence‐activated cell sorting to identify mutants that bound to α_vβ₃ integrin. Select peptides were synthesized and were shown to compete for natural ligand binding to integrin receptors expressed on the surface of U87MG glioblastoma cells with half‐maximal inhibitory concentration values of 10–30 nM. Receptor specificity assays demonstrated that engineered knottin peptides bind to both α_vβ₃ and α_vβ₅ integrins with high affinity. Interestingly, we also discovered a peptide that binds with high affinity to α_vβ₃, α_vβ₅, and α₅β₁ integrins. This finding has important clinical implications because all three of these receptors can be coexpressed on tumors. In addition, we showed that engineered knottin peptides inhibit tumor cell adhesion to the extracellular matrix protein vitronectin, and in some cases fibronectin, depending on their integrin binding specificity. Collectively, these data validate EETI‐II as a scaffold for protein engineering, and highlight the development of unique integrin‐binding peptides with potential for translational applications in cancer. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Conformational constraints in angiotensin IV to probe the role of Tyr2, Pro5 and Phe6

The aromatic amino acids Tyr and Phe in angiotensin IV (Ang IV) were conformationally constrained by the use of β‐Me substituted analogs, or cyclic constrained analogs. None of these modifications was allowed for Tyr¹, while only e‐β‐MePhe⁶ substitution resulted in an AngIV analog with high IRAP potency and selectivity versus AP‐N or the AT₁ receptor. This indicates an important role of the orientation of the Phe⁶ for inducing selectivity. Pro⁵ replacement with 2‐aminocyclopentanecarboxylic acid maintained IRAP potency and abolished AT₁ affinity. These results confirm the importance of conformational constrained amino acids to generate selectivity in bioactive peptides. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

TAPP analogs containing β3‐homo‐amino acids: synthesis and receptor binding

β‐Amino acids containing α,β‐hybrid peptides show great potential as peptidomimetics. In this paper, we describe the synthesis and affinity to μ‐opioid and δ‐opioid receptors of α,β‐hybrids, analogs of the tetrapeptide Tyr‐ d ‐Ala‐Phe‐Phe‐NH₂ (TAPP). Each amino acid was replaced with an l ‐ or d ‐β³‐h‐amino acid. All α,β‐hybrids of TAPP analogs were synthesized in solution and tested for affinity to μ‐opioid and δ‐opioid receptors. The analog Tyr‐β³h‐ d ‐Ala‐Phe‐PheNH₂ was found to be as active as the native tetrapeptide. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

A naturally occurring αs1‐casein‐derived peptide in bovine milk inhibits apoptosis of granulosa cells induced by serum‐free conditions

Several naturally occurring peptides in bovine milk were characterized by tandem mass spectrometry and Edman degradation. Chromatograms of peptide fractions (passed through an ultra‐filtration membrane, nominal molecular weight limit 3000) prepared from colostrum (collected immediately after parturition) and transitional milk (collected 5 days postpartum) showed that they were almost identical. In total, six peptides, α_s1‐CN (f16‐23) (RPKHPIKH), α_s1‐CN (f16‐24) (RPKHPIKHQ), α_s1‐CN (f17‐25) (PKHPIKHQG), α_s1‐CN (f46‐52) (VFGKEKV), α_s1‐CN (f94‐105) (HIQKEDVPSER), and β‐CN (f121‐128) (HKEMPFPK), were identified. One of the major peptides, the N‐terminal fragment of α_s1‐casein, varied structurally during early lactation: α_s1‐CN (f17‐25) (PKHPIKHQG) and α_s1‐CN (f16‐23) (RPKHPIKH)/α_s1‐CN (f16‐24) (RPKHPIKHQ) were found in colostrum and transitional milk, respectively. A chemically synthesized peptide, α_s1‐CN (f16‐23) (RPKHPIKH), inhibited apoptosis of bovine granulosa cells induced by serum‐free conditions in a dose‐dependent manner, in consequence of caspase‐3 and caspase‐9 suppressions. The physiological function of the peptide remains unclear, but it may have potential use as pharmaceutical agent and as an anti‐apoptotic agent in cell culture medium. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Structure primaire de l'anhydrase carbonique érythrocytaire B humaine: II. Clivage par le bromure de cyanogène et séquence des résidus 1–148

The amino acid sequence of the IICNBr fragment of the human erythrocyte carbonic anhydrase B has been determined. This fragment contains the first 148 of the 260 residues of the N-acetylated single polypeptide chain of the protein. After tryptic hydrolysis of this fragment, eleven peptides have been isolated by gel filtration and chromatography on Dowex 50 W-X₂ or DEAE-Sephadex. Eight of them were identified with already sequenced peptides previously isolated from tryptic hydrolysate of the whole protein. The other three ones were obtained in pure form and sequenced. The combined amino acid content of these eleven peptides only account for 124 of the 148 amino acid residues in the IICNBr fragment. The tryptic attack of the maleylated IICNBr fragment gave three peptides as was expected from the number of arginine residues (2) in this fragment: two arginyl peptides (II₁, II₃) and one homoseryl peptide (II₂). They were purified by gel filtration. The unidentified 24 residue tryptic peptide has been isolated from the demaleylated II₂ tryptic hydrolysate and sequenced. The order of the twelve tryptic peptides of IICNBr fragment has been obtained by study of chymotrypsic peptides isolated from II₁ and IICNBr fragment.     

Bh3 induced conformational changes in Bcl‐Xl revealed by crystal structure and comparative analysis

Apoptosis or programmed cell death is a regulatory process in cells in response to stimuli perturbing physiological conditions. The Bcl‐2 family of proteins plays an important role in regulating homeostasis during apoptosis. In the process, the molecular interactions among the three members of this family, the pro‐apoptotic, anti‐apoptotic and BH3‐only proteins at the mitochondrial outer membrane define the fate of a cell. Here, we report the crystal structures of the human anti‐apoptotic protein Bcl‐X_L in complex with BH3‐only BID^BH3 and BIM^BH3 peptides determined at 2.0 Å and 1.5 Å resolution, respectively. The BH3 peptides bind to the canonical hydrophobic pocket in Bcl‐X_L and adopt an alpha helical conformation in the bound form. Despite a similar structural fold, a comparison with other BH3 complexes revealed structural differences due to their sequence variations. In the Bcl‐X_L‐BID^BH3 complex we observed a large pocket, in comparison with other BH3 complexes, lined by residues from helices α1, α2, α3, and α5 located adjacent to the canonical hydrophobic pocket. These results suggest that there are differences in the mode of interactions by the BH3 peptides that may translate into functional differences in apoptotic regulation. Proteins 2015; 83:1262–1272. © 2015 Wiley Periodicals, Inc.     

A self T cell epitope induces autoantibody response: mechanism for production of antibodies to diverse glomerular basement membrane antigens

The anti-glomerular basement membrane (GBM) Ab has been regarded as a prototypical example of pathogenic autoantibodies. However, the mechanism for elicitation of this Ab remains unknown. In the present paper, we report that the Ab to diverse GBM Ags was induced by a single nephritogenic T cell epitope in a rat model. The T cell epitope pCol(28-40) of noncollagen domain 1 of collagen type IV alpha3 chain not only uniformly induced severe glomerulonephritis but also elicited anti-GBM Ab in 76% of the immunized rats after prominent glomerular injury. Furthermore, we demonstrated that the anti-GBM Ab was not related to the peptidic B cell epitope nested in pCol(28-40); that is, 1) elimination of the B cell epitope, either by substitution of the critical residues of the B cell epitope or by truncation, failed to abrogate anti-GBM Ab production, and 2) the anti-GBM Ab, eluted from the diseased kidneys, reacted only with native GBM, but not with pCol(28-40). Confocal microscopy and immunoprecipitation further demonstrated that the eluted anti-GBM Ab recognized conformational B cell epitope(s) of multiple native GBM proteins. We conclude that autoantibody response to diverse native GBM Ags was induced by a single nephritogenic T cell epitope. Thus, anti-GBM Ab may actually be a consequence of T cell-mediated glomerulonephritis.     

Mutational analysis of type IV collagen alpha5 chain, with respect to heterotrimer formation

Alport syndrome (AS) is caused by mutations in type IV collagen α3, α4, and α5 chains. The three chains form a heterotrimer. In this study, we introduced 12 kinds of missense and three kinds of nonsense mutations, corresponding to AS mutations, into the NC1 domain of α5(IV) and characterized the mutant chains. Nine α5(IV) chains with amino acid substitutions and all three truncated α5(IV) chains did not form a heterotrimer and were not secreted from cells. Three α5(IV) chains with amino acid substitutions did, however, form heterotrimers in cells, but these were not secreted from cells. These findings indicate that a defect in heterotrimer formation is the main molecular mechanism underlying the pathogenesis of AS caused by mutation in the NC1 domain. We also showed that even a single amino acid deletion in the carboxyl-terminal region markedly affected the heterotrimerization, indicating that the carboxyl-terminal end is indispensable for heterotrimer formation.     

The goodpasture autoantigen. Mapping the major conformational epitope(s) of alpha3(IV) collagen to residues 17-31 and 127-141 of the NC1 domain

The Goodpasture (GP) autoantigen has been identified as the alpha3(IV) collagen chain, one of six homologous chains designated alpha1-alpha6 that comprise type IV collagen (Hudson, B. G., Reeders, S. T., and Tryggvason, K. (1993) J. Biol. Chem. 268, 26033-26036). In this study, chimeric proteins were used to map the location of the major conformational, disulfide bond-dependent GP autoepitope(s) that has been previously localized to the noncollagenous (NC1) domain of alpha3(IV) chain. Fourteen alpha1/alpha3 NC1 chimeras were constructed by substituting one or more short sequences of alpha3(IV)NC1 at the corresponding positions in the non-immunoreactive alpha1(IV)NC1 domain and expressed in mammalian cells for proper folding. The interaction between the chimeras and eight GP sera was assessed by both direct and inhibition enzyme-linked immunosorbent assay. Two chimeras, C2 containing residues 17-31 of alpha3(IV)NC1 and C6 containing residues 127-141 of alpha3(IV)NC1, bound autoantibodies, as did combination chimeras containing these regions. The epitope(s) that encompasses these sequences is immunodominant, showing strong reactivity with all GP sera and accounting for 50-90% of the autoantibody reactivity toward alpha3(IV)NC1. The conformational nature of the epitope(s) in the C2 and C6 chimeras was established by reduction of the disulfide bonds and by PEPSCAN analysis of overlapping 12-mer peptides derived from alpha1- and alpha3(IV)NC1 sequences. The amino acid sequences 17-31 and 127-141 in alpha3(IV)NC1 have thus been shown to contain the critical residues of one or two disulfide bond-dependent conformational autoepitopes that bind GP autoantibodies.     

Identification of Critical Residues of Linear B Cell Epitope on Goodpasture Autoantigen

Background

The autoantigen of anti-glomerular basement membrane (GBM) disease has been identified as the non-collagenous domain 1 of α3 chain of type IV collagen, α3(IV)NC1. Our previous study revealed a peptide on α3(IV)NC1 as a major linear epitope for B cells and potentially nephrogenic, designated as P14 (α3129-150). This peptide has also been proven to be the epitope of auto-reactive T cells in anti-GBM patients. This study was aimed to further characterize the critical motif of P14.

Methods

16 patients with anti-GBM disease and positive anti-P14 antibodies were enrolled. A set of truncated and alanine substituted peptides derived from P14 were synthesized. Circulating antibodies against the peptides were detected by enzyme linked immunosorbent assay (ELISA).

Results

We found that all sera with anti-P14 antibodies reacted with the 13-mer sequence in the C-terminus of P14 (P14c) exclusively. The level of antibodies against P14 was highly correlated with the level of antibodies against P14c (r=0.970, P<0.001). P14c was the core immunogenic region and the amino acid sequence (ISLWKGFSFIMFT) was highly hydrophobic. Each amino acid residue in P14c was sequentially replaced by alanine. Three residues of glycine142, phenylalanine143, and phenylalanine145 were identified crucial for antibody binding based on the remarkable decline (P<0.001) of antibody reaction after each residue replacement.

Conclusions

We defined GFxF (α3142, 143,145) as the critical motif of P14. It may provide some clues for understanding the etiology of anti-GBM disease.     

Enhanced vasoconstriction to α_1 adrenoceptor autoantibody in spontaneously hypertensive rats

Autoimmune activities have been implicated in the pathogenesis of hypertension.High levels of autoantibodies against the second extracellular loop of α1-adrenoceptor(α1-AR autoantibody,α1-AA) are found in patients with hypertension,and α1-AA could exert a α1-AR agonist-like vasoconstrictive effect.However,whether the vasoconstrictive effect of α1-AA is enhanced in hypertension is unknown.Using aortic rings of spontaneously hypertensive rats(SHR) and normotensive Wistar-Kyoto(WKY) rats,we observed the vasoconstrictive responses to α1-AA with phenylephrine(α1-AR agonist) as a positive control drug.Aortic nitrotyrosine levels were also measured by ELISA and immunohistochemistry.The results showed that the aortic constrictive responses to α1-AA and phenylephrine(both 1 nmol L-1-10 μmol L-1) were greater in SHR than in WKY rats.Endothelial denudation or L-NAME(a non-selective NOS inhibitor)(100 μmol L-1) increased α1-AA- or phenylephrine-induced vasoconstrictions both in SHR and WKY.However,selective iNOS inhibitor 1400W(10 μmol L-1) enhanced the α1-AA-induced aortic constriction in WKY,but not in SHR.The aortic nitrotyrosine level was significantly higher in SHR than WKY,as shown by both ELISA and immunohistochemistry.These results indicate that the vasoconstrictive response to α1-AA is enhanced in SHR,and this altered responsiveness is due to endothelial dysfunction and decreased NO bioavailability.The study suggests an important role of α1-AR autoimmunity in the pathogenesis and management of hypertension especially in those harboring high α1-AA levels.     

Epitope analysis of the multiphosphorylated peptide αs1‐casein(59–79)

The multiphosphorylated tryptic peptide α_s1‐casein(59–79) has been shown to be antigenic with anti‐casein antibodies. In an approach to determine the amino acyl residues critical for antibody binding we undertook an epitope analysis of the peptide using overlapping synthetic peptides. With α_s1‐casein(59–79) as the adsorbed antigen in a competitive ELISA only two of five overlapping synthetic peptides at 1 mM significantly inhibited binding of the anti‐casein antibodies. Peptides Glu‐Ser(P)‐Ile‐Ser(P)‐Ser(P)‐Ser(P)‐Glu‐Glu and Ile‐Val‐Pro‐Asn‐Ser(P)‐Val‐Glu‐Glu inhibited antibody binding by 20.0±3.6% and 60.3±7.9%, respectively. The epitope of Glu⁶³‐Ser(P)‐Ile‐Ser(P)‐Ser(P)‐Ser(P)‐Glu‐Glu⁷⁰ was further localised to the phosphoseryl cluster as the peptide Ser(P)‐Ser(P)‐Ser(P) significantly inhibited binding of the anti‐casein antibodies to α_s1‐casein(59–79) by 29.5±7.4%. Substitution of Ser(P)⁷⁵ with Ser⁷⁵ in the second inhibitory peptide Ile‐Val‐Pro‐Asn‐Ser(P)⁷⁵‐Val‐Glu‐Glu also abolished inhibition of antibody binding to α_s1‐casein (59–79) demonstrating that Ser(P)⁷⁵ is also a critical residue for recognition by the antibodies. These data show that the phosphorylated residues in the cluster sequence ‐Ser(P)⁶⁶‐Ser(P)‐Ser(P)⁶⁸ and in the sequence ‐Pro⁷³‐Asn‐Ser(P)‐Val‐Glu⁷⁷‐ are critical for antibody binding to α_s1‐casein(59–79) and further demonstrate that a highly phosphorylated segment of a protein can be antigenic. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

In vitro synthesis of the Bowman-Birk and related soybean protease inhibitors

Poly (A)⁺ RNAs from immature soybean seeds were size fractionated in denaturing sucrose gradients to identify mRNA that directs the cell-free synthesis of the Bowman-Birk protease inhibitor and the related inhibitors PI I–IV. Polypeptides synthesized in vitro were labeled with (³⁵S)-cysteine and (³H)-serine and detected by immunoprecipitation with anti Bowman-Birk and anti PI I–IV sera. Immunoprecipitates of the translation products comigrated on SDS-polyacrylamide gels with the dimeric or trimeric aggregates of the authentic inhibitor proteins, which self-associate under certain conditions. Further evidence that these immunoprecipitates contained authentic polypeptides corresponding to the Bowman-Birk or PI IV inhibitor was shown by sequential amino acid analyses of peptides generated by cleavage with cyanogen bromide.