A spatial similarity of stereochemical environments formed by amino acid residues defines a common epitope of two non-homologous proteins

Abstract It is critical for development of high-quality antibodies in research and diagnostics to predict accurately their cross-reactivities with “off-target” molecules, which potentially induce false results. Herein, we report a good example of such a cross-reactivity for an off-target due to a stereochemical environment of epitopes, which does not simply depend on amino acid sequences. We found that significant subpopulation of a polyclonal peptide antibody against Bcnt (Bucentaur) (anti-BCNT-C antibody) cross-reacted with a completely different protein, glutamine synthetase (GS), and identified four amino acids, GYFE, in its C-terminal region as the core amino acids for the cross-reaction. Consistent with this finding, the anti-BCNT-C antibody strongly recognized endogenously and exogenously expressed GS in tissues and cultured cells by Western blotting and immunohistochemistry. Furthermore, we elucidated that the cross-reaction is caused by a spatial similarity between the stereochemical environments formed by amino acid residues, including the GYFE of GS and the GYIE of Bcnt, rather than by their primary sequences. These results suggest it is critical to comprehensively analyze antibody interactions with target molecules including off-targets with special attention to the physicochemical environments of epitope-paratope interfaces to decrease the risk of false interpretations of results using antibodies in science and clinical applications.

Download Full-text

Characterization and Construction of Functional cDNA Clones of Pariacoto Virus, the First Alphanodavirus Isolated outside Australasia

Journal of Virology ◽

10.1128/jvi.74.11.5123-5132.2000 ◽

2000 ◽

Vol 74 (11) ◽

pp. 5123-5132 ◽

Cited By ~ 60

Author(s):

Karyn N. Johnson ◽

Jean-Louis Zeddam ◽

L. Andrew Ball

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Capsid Protein ◽

Cultured Cells ◽

Geographic Origin ◽

Amino Acid Sequences ◽

Cdna Clones ◽

Homologous Proteins ◽

Genomic Rnas ◽

Rna Genome

ABSTRACT Pariacoto virus (PaV) was recently isolated in Peru from the Southern armyworm (Spodoptera eridania). PaV particles are isometric, nonenveloped, and about 30 nm in diameter. The virus has a bipartite RNA genome and a single major capsid protein with a molecular mass of 39.0 kDa, features that support its classification as aNodavirus. As such, PaV is the firstAlphanodavirus to have been isolated from outside Australasia. Here we report that PaV replicates in wax moth larvae and that PaV genomic RNAs replicate when transfected into cultured baby hamster kidney cells. The complete nucleotide sequences of both segments of the bipartite RNA genome were determined. The larger genome segment, RNA1, is 3,011 nucleotides long and contains a 973-amino-acid open reading frame (ORF) encoding protein A, the viral contribution to the RNA replicase. During replication, a 414-nucleotide long subgenomic RNA (RNA3) is synthesized which is coterminal with the 3′ end of RNA1. RNA3 contains a small ORF which could encode a protein of 90 amino acids similar to the B2 protein of other alphanodaviruses. RNA2 contains 1,311 nucleotides and encodes the 401 amino acids of the capsid protein precursor α. The amino acid sequences of the PaV capsid protein and the replicase subunit share 41 and 26% identity with homologous proteins of Flock house virus, the best characterized of the alphanodaviruses. These and other sequence comparisons indicate that PaV is evolutionarily the most distant of the alphanodaviruses described to date, consistent with its novel geographic origin. Although the PaV capsid precursor is cleaved into the two mature capsid proteins β and γ, the amino acid sequence at the cleavage site, which is Asn/Ala in all other alphanodaviruses, is Asn/Ser in PaV. To facilitate the investigation of PaV replication in cultured cells, we constructed plasmids that transcribed full-length PaV RNAs with authentic 5′ and 3′ termini. Transcription of these plasmids in cells recreated the replication of PaV RNA1 and RNA2, synthesis of subgenomic RNA3, and translation of viral proteins A and α.

Download Full-text

Nucleotide and derived amino acid sequences of a cDNA coding for pre-uteroglobin from the lung of the hare (Lepus capensis)

Biochemical Journal ◽

10.1042/bj2350895 ◽

1986 ◽

Vol 235 (3) ◽

pp. 895-898 ◽

Cited By ~ 12

Author(s):

M S López de Haro ◽

A Nieto

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Nucleotide Sequence ◽

Amino Acid Sequence ◽

Amino Acid Sequences ◽

Untranslated Regions ◽

Coding Region ◽

Homologous Proteins ◽

Lepus Capensis ◽

Rabbit Gene

An almost full-length cDNA coding for pre-uteroglobin from hare lung was cloned and sequenced. The derived amino acid sequence indicated that hare pre-uteroglobin contained 91 amino acids, including a signal peptide of 21 residues. Comparison of the nucleotide sequence of hare pre-uteroglobin cDNA with that previously reported for the rabbit gene indicated five silent point substitutions and six others leading to amino acid changes in the coding region. The untranslated regions of both pre-uteroglobin mRNAs were very similar. The amino acid changes observed are discussed in relation to the different progesterone-binding abilities of both homologous proteins.

Download Full-text

Targeting motifs and functional parameters governing the assembly of connexins into gap junctions

Biochemical Journal ◽

10.1042/bj3490281 ◽

2000 ◽

Vol 349 (1) ◽

pp. 281-287 ◽

Cited By ~ 7

Author(s):

Patricia E. M. MARTIN ◽

James STEGGLES ◽

Claire WILSON ◽

Shoeb AHMAD ◽

W. Howard EVANS

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Gap Junctions ◽

Gap Junction ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Lucifer Yellow ◽

Amino Acid Sequences ◽

Amino Acid Residues ◽

Green Fluorescent

To study the assembly of gap junctions, connexin-green-fluorescent-protein (Cx-GFP) chimeras were expressed in COS-7 and HeLa cells. Cx26- and Cx32-GFP were targeted to gap junctions where they formed functional channels that transferred Lucifer Yellow. A series of Cx32-GFP chimeras, truncated from the C-terminal cytoplasmic tail, were studied to identify amino acid sequences governing targeting from intracellular assembly sites to the gap junction. Extensive truncation of Cx32 resulted in failure to integrate into membranes. Truncation of Cx32 to residue 207, corresponding to removal of most of the 78 amino acids on the cytoplasmic C-terminal tail, led to arrest in the endoplasmic reticulum and incomplete oligomerization. However, truncation to amino acid 219 did not impair Cx oligomerization and connexon hemichannels were targeted to the plasma membrane. It was concluded that a crucial gap-junction targeting sequence resides between amino acid residues 207 and 219 on the cytoplasmic C-terminal tail of Cx32. Studies of a Cx32E208K mutation identified this as one of the key amino acids dictating targeting to the gap junction, although oligomerization of this site-specific mutation into hexameric hemichannels was relatively unimpaired. The studies show that expression of these Cx-GFP constructs in mammalian cells allowed an analysis of amino acid residues involved in gap-junction assembly.

Download Full-text

Two Highly Similar Chitinases from Marine Vibrio Species have Different Enzymatic Properties

Marine Drugs ◽

10.3390/md18030139 ◽

2020 ◽

Vol 18 (3) ◽

pp. 139

Author(s):

Xinxin He ◽

Min Yu ◽

Yanhong Wu ◽

Lingman Ran ◽

Weizhi Liu ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Extracellular Enzymes ◽

Vibrio Harveyi ◽

Amino Acid Sequences ◽

Enzymatic Properties ◽

Site Directed Mutagenesis ◽

Amino Acid Residues ◽

Marine Vibrio ◽

Key Sites

Chitinase, as one of the most important extracellular enzymes in the marine environment, has great ecological and applied values. In this study, two chitinases (Chi1557 and Chi4668) with 97.33% amino acid sequences identity were individually found in Vibrio rotiferianus and Vibrio harveyi. They both were encoding by 561 amino acids, but differed in 15 amino acids and showed different enzymatic properties. The optimal temperature and pH ranges were 45–50 °C and pH 5.0–7.0 for Chi1557, while ~50 °C and pH 3.0–6.0 for Chi4668. K+, Mg2+, and EDTA increased the enzymatic activity of Chi4668 significantly, yet these factors were inhibitory to Chi1557. Moreover, Chi1557 degraded colloidal chitin to produce (GlcNAc)2 and minor GlcNAc, whereas Chi4668 produce (GlcNAc)2 with minor (GlcNAc)3 and (GlcNAc)4. The Kcat/Km of Chi4668 was ~4.7 times higher than that of Chi1557, indicating that Chi4668 had stronger catalytic activity than Chi1557. Furthermore, site-directed mutagenesis was performed on Chi1557 focusing on seven conserved amino acid residues of family GH18 chitinases. Chi1557 was almost completely inactive after Glu154, Gln219, Tyr221, or Trp312 was individually mutated, retained ~50% activity after Tyr37 was mutated, and increased two times activity after Asp152 was mutated, indicating that these six amino acids were key sites for Chi1557.

Download Full-text

Molecular Cloning and Bioinformatics Analysis of DQA Gene from Mink (Neovison vison)

International Journal of Molecular Sciences ◽

10.3390/ijms20051037 ◽

2019 ◽

Vol 20 (5) ◽

pp. 1037

Author(s):

Zhaobin Fan ◽

Houfeng Zhang ◽

Min Rong ◽

Dongmei Meng ◽

Zhenxing Yu ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Molecular Mechanisms ◽

Signal Sequence ◽

Amino Acid Sequences ◽

Neovison Vison ◽

Random Coil ◽

Amino Acid Residues ◽

Coding Region ◽

Full Length Sequence

In the present study, we cloned, sequenced, and explored the structural and functional characteristics of the major histocompatibility complex (MHC)-DQA gene from mink (Neovison vison) for the first time. The full-length sequence of DQA gene was 1147-bp-long, contained a coding region of 768-bp, which was predicted to encoding 255 amino acid residues. The comparison between DQA from mink (Neovison vison) and other MHC-DQA molecules from different animal species showed that nucleotide and encoded amino acid sequences of the mink DQA gene exhibited high similarity with the ferret (Mustela pulourius furo). Phylogenetic analysis revealed that mink (Neovison vison) DQA is grouped with that of ferret (Mustela pulourius furo). The cloned sequence contained a 23-amino acid NH2-terminal signal sequence with the signal peptide cutting site located in amino acids 23–24, and had three Asn-Xaa-Ser/Thr sequons. Three cysteine residues were also identified (Cys-85, Cys-121, and Cys-138). The 218 to 240 amino acids were predicted to be the transmembrane domains. The prediction of the secondary structure revealed three α-helixes and fourteen β-sheets in Neovison vison DQA protein, while random coil was a major pattern. In this study, the whole CDS sequence of Neovison vison DQA gene was successfully cloned, which was valuable for exploring the function and antiviral molecular mechanisms underlying the molecule. The findings of the present study have laid the foundation for the disease resistance and breeding of mink.

Download Full-text

Amino acid sequences C-terminal to the cross-links in bovine elastin

Biochemical Journal ◽

10.1042/bj1850611 ◽

1980 ◽

Vol 185 (3) ◽

pp. 611-616 ◽

Cited By ~ 16

Author(s):

K M Baig ◽

M Vlaovic ◽

R A Anwar

Keyword(s):

Amino Acid ◽

Amino Acid Sequences ◽

Amino Acid Residues ◽

Tyrosine Residues ◽

Lysine Residues ◽

Cross Links ◽

The Cross

All the desmosine-containing elastolytic peptides of bovine ligamentum-nuchae elastin have now been examined for amino acid sequences C-terminal to the cross-links. In addition, amino acid residues C-terminal to lysine residues in bovine tropoelastin were also examined. No tyrosine C-terminal to cross-links in bovine elastin or C-terminal to lysine in tropoelastin was detected. Apparently all the tyrosine residues C-terminal to lysine residues in pig tropoelastin are replaced with phenylalanine in bovine tropoelastin. All the data presented are consistent with the scheme proposed for the formation of desmosine and isodesmosine cross-links of elastin by Gerber & Anwar [(1975) Biochem. J. 149, 685-695].

Download Full-text

Author Correction: A spatial similarity of stereochemical environments formed by amino acid residues defines a common epitope of two non-homologous proteins

Scientific Reports ◽

10.1038/s41598-020-68279-6 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Kentaro Nakashima ◽

Shintaro Iwashita ◽

Takehiro Suzuki ◽

Chieko Kato ◽

Toshiyuki Kohno ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Residues ◽

Homologous Proteins ◽

Spatial Similarity

Download Full-text

The Amino Acid Sequence of Streptomyces griseus Protease A: The Peptic Peptides

Canadian Journal of Biochemistry ◽

10.1139/o71-158 ◽

1971 ◽

Vol 49 (10) ◽

pp. 1083-1097 ◽

Cited By ~ 13

Author(s):

P. Johnson ◽

L. B. Smillie

Keyword(s):

Amino Acid ◽

Streptomyces Griseus ◽

Disulfide Bridge ◽

Exchange Chromatography ◽

Amino Acid Sequences ◽

Amino Acid Residues ◽

Homologous Proteins ◽

Acidic Peptide ◽

Dowex 1 ◽

Peptide Fractions

The peptic peptides of Streptomyces griseus Protease A (excluding the previously characterized disulfide bridge peptic peptides) were fractionated into basic, neutral, and neutral plus acidic peptide fractions by chromatography on Dowex 1 × 2. These three peptide fractions were then fractionated by cation-exchange chromatography on Chromobead P resin using the Technicon autoanalyzer system. Following further purifications on paper, the amino acid compositions and sequences of the peptic peptides were determined. The N-terminal sequence of Protease A has been identified as Ile–Ala–Gly–Gly–Glu–Ala. The numbers of amino acid residues obtained from the amino acid sequences reported are in agreement with those numbers obtained from amino acid analysis of the total protein in the cases of tryptophan, methionine, histidine, proline, phenylalanine, and glutamic acid. Some of the results suggest either the presence of nonidentical but highly homologous proteins in the Protease A preparation or the possibility of repeating sequences in a single molecular species.

Download Full-text

Molecular cloning and sequence analysis of the cDNA for ancrod, a thrombin-like enzyme from the venom of Calloselasma rhodostoma

Biochemical Journal ◽

10.1042/bj2940387 ◽

1993 ◽

Vol 294 (2) ◽

pp. 387-390 ◽

Cited By ~ 46

Author(s):

L C Au ◽

S B Lin ◽

J S Chou ◽

G W Teh ◽

K J Chang ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Serine Proteases ◽

Sequence Similarity ◽

Active Form ◽

Amino Acid Sequences ◽

Amino Acid Residues ◽

Binding Reaction ◽

Venom Glands ◽

High Degree

The 1.54 kb cDNA for ancrod, a thrombin-like enzyme, was cloned from a lambda ZAP cDNA library derived from the venom glands of Calloselasma (Agkistrodon) rhodostoma. The cDNA sequence reveals that ancrod is synthesized as a pre-zymogen of 258 amino acids, including a putative secretory peptide of 18 amino acids and a proposed zymogen peptide of 6 amino-acid residues. The amino-acid sequence of the predicted active form of the enzyme exhibits a high degree of sequence similarity to those of mammalian serine proteases (trypsin and pancreatic kallikrein) and other thrombin-like enzymes (batroxobin and flavoxobin). Key amino-acid residues (His43, Asp88, Ser182 and Asp176) that are thought to be involved in the substrate cleavage and in the substrate-binding reaction are conserved. Ancrod contains 13 cysteine residues. Based on alignment with the amino-acid sequences of trypsin and batroxobin, six disulphide bridges can be predicted to be present in the ancrod protein. The existence of a free cysteine, which changes the common sequence surrounding the Ser182 active site from Gly-Asp-Ser-Gly-Gly-Pro to Cys-Asp-Ser-Gly-Gly-Pro, is unusual for a serine protease.

Download Full-text

Comprehensive mutagenesis to identify amino acid residues contributing to the difference in thermostability between two originally thermostable ancestral proteins

PLoS ONE ◽

10.1371/journal.pone.0258821 ◽

2021 ◽

Vol 16 (10) ◽

pp. e0258821

Author(s):

Satoshi Akanuma ◽

Minako Yamaguchi ◽

Akihiko Yamagishi

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Rational Design ◽

Synergistic Effects ◽

Amino Acid Sequences ◽

Amino Acid Substitutions ◽

Amino Acid Residues ◽

Identify Amino Acid ◽

The Difference ◽

The Stability

Further improvement of the thermostability of inherently thermostable proteins is an attractive challenge because more thermostable proteins are industrially more useful and serve as better scaffolds for protein engineering. To establish guidelines that can be applied for the rational design of hyperthermostable proteins, we compared the amino acid sequences of two ancestral nucleoside diphosphate kinases, Arc1 and Bac1, reconstructed in our previous study. Although Bac1 is a thermostable protein whose unfolding temperature is around 100°C, Arc1 is much more thermostable with an unfolding temperature of 114°C. However, only 12 out of 139 amino acids are different between the two sequences. In this study, one or a combination of amino acid(s) in Bac1 was/were substituted by a residue(s) found in Arc1 at the same position(s). The best mutant, which contained three amino acid substitutions (S108D, G116A and L120P substitutions), showed an unfolding temperature more than 10°C higher than that of Bac1. Furthermore, a combination of the other nine amino acid substitutions also led to improved thermostability of Bac1, although the effects of individual substitutions were small. Therefore, not only the sum of the contributions of individual amino acids, but also the synergistic effects of multiple amino acids are deeply involved in the stability of a hyperthermostable protein. Such insights will be helpful for future rational design of hyperthermostable proteins.

Download Full-text