Immune Rejection Mediated by prf1 and gzmb Affects the Colonization of Fat Greenling (Hexagrammos otakii) Spermatogonia in Heterotransplantation.

Fish germ cell transplantation holds great potential for conserving endangered species, improving cultured fish breeds, and exploring reproductive techniques. However, low transplantation efficiency is a common issue in heterotransplantation. This study transplanted fat greenling (Hexagrammos otakii) spermatogonia into the testes of spotted sea bass (Lateolabrax maculatus) to investigate factors that might affect the colonization and fixation of heterologous transplanted germ cells. Results indicated that transplanted fat greenling spermatogonia cells were successfully detected in the early transplantation phase in spotted sea bass. Their numbers gradually decreased over time, and after 10 days post-transplantation, more than 90% of the transplanted cells underwent apoptosis. Transcriptome sequencing analysis of the testes of spotted sea bass and fat greenling spermatogonia on days 1 and 10 post-transplantation revealed that this apoptosis process involved many immune-related genes and their associated signaling pathways. Acute immune rejection marker genes prf1 and gzmb were detected in the spotted sea bass testes, while immune tolerance genes lck and zap-70 were expressed in the fat greenling spermatogonia. Additionally, differential expression of prf1 and gzmb genes was screened from spotted sea bass, with experimental evidence indicating that PRF1 and GZMB protein from spotted sea bass primarily induce apoptosis in transplanted fat greenling spermatogonia via the mitochondrial apoptosis pathway, at the protein level. This suggests that the difficulties in heterotransplantation are primarily related to acute immune rejection, with PRF1 and GZMB playing significant roles.

Structural insights revealed by crystal structure of B38-CAP, an isoenzyme of carboxypeptidase ACE2, the receptor of SARS-CoV-2.

The worldwide pandemic of Coronavirus disease 2019 (COVID-19) is triggered by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and further worsened by the emergence of a variety of SARS-CoV-2 variants. Angiotensin-converting enzyme 2 (ACE2), a carboxypeptidase of M32 family, serves as the receptor of SARS-CoV-2 and key regulator of host renin-angiotensin system (RAS), both of which are mainly mediated via the carboxypeptidase domain of ACE2 (sACE2) or its activity. sACE2 is thus promising in the treatment of COVID-19 but unfortunately weakened by its unstrigent substrate preference and complex interplay with host RAS. B38-CAP, an isoenzyme of ACE2, partically compensates these defects but still encounters the problem related to carboxypeptidase activity and specificity. In this study, we firstly determined the crystal structure of B38-CAP at a resolution of 2.44 Å which exists in dimeric form with the non-crystallographic two-fold axis being in coincidence with the crystallographic two-fold axis. Further structural analysis revealed the structural conservatism feature among M32 family, particularly the catalytic core and moreover lead us to hypothesize that conformational flexibility might play an pivotal role in the catalysis of B38-CAP and ACE2. The work provided here presents key features of the M32 family carboxypeptidase and provides structural basis for further development of B38-CAP-based anti-SARS-CoV-2 drugs.

Plant endophytic bacteria: A potential resource pool of electroactive micro-organisms.

AIMS: Electroactive micro-organisms play a significant role in microbial fuel cells. It is necessary to discover potential resources in plant endophytes. In this study, plant tissues were selected to isolate endophytic bacteria, and the electrochemical activity potential was evaluated. METHODS AND RESULTS: The microbial fuel cell (MFC) is used to evaluate the electricity-producing activity of endophytic bacteria in plant tissues, and the species distribution of micro-organisms in the anode of the MFC after inoculation of plant tissues is determined by high-throughput sequencing. Twenty-six strains of bacteria were isolated from plant tissues belonging to Angelica and Sweet Potato, of which 17 strains from six genera had electrochemical activity, including Bacillus sp., Pleomorphomonas sp., Rahnella sp., Shinella sp., Paenibacillus sp. and Staphylococcus sp. Moreover, the electricity-producing micro-organisms in the plant tissue are enriched. Pseudomonas and Clostridioides are the dominant genera of MFC anode inoculated with angelica tissue. Staphylococcus and Lachnoclostridium are the dominant genera in MFC anode inoculated with sweet potato tissue. And the most representative Gram-positive strain Staphylococcus succinus subsp. succinus H6 and plant tissue were further analysed for electrochemical activity. And a strain numbered H6 and plant tissue had a good electrogenerating activity. CONCLUSION: This study is of great significance for expanding the resource pool of electricity-producing micro-organisms and tapping the potential of plant endophytes for electricity-producing. SIGNIFICANCE AND IMPACT OF STUDY: This is the first study to apply plant endophytes to MFC to explore the characteristics of electricity production. It is of great significance for exploring the diversity of plant endophytes and the relationship between electricity producing bacteria and plants.

Isolation, Identification and Characteristic Analysis of Plant Endophyte Electrogenic Bacteria Shinella zoogloeoides SHE10.

Electroactive microorganisms play a significant role in microbial fuel cells (MFCs). These devices are environmentally friendly and can turn large quantities of organic material into renewable energy based on microbial diversity. Based on broad microbial diversity, it is necessary to obtain a comprehensive understanding of their resource distribution and to discover potential resources. In this study, sweet potato tissues were selected to isolate endophytic bacteria, and the electrochemical activity potential of those bacteria was evaluated by high-throughput screening with a WO3 nanoprobe. This study was screened and obtained a strain SHE10 with electrochemical performance from the rhizome of sweet potato by a WO3 nanoprobe, which was identified as Shinella zoogloeoides. After nearly 600 h of voltage monitoring and cyclic voltammetry analysis, the results showed that the average voltage of S. zoogloeoides SHE10 reached 122.5 mV in stationary period. The maximum power density is 78.3 ± 1.8 mW/m2, and the corresponding current density is 223.0 mA/m2. The good redox reaction also indicated that the strain had good electrical activity. Its electron transfer mode was diverse, but its power generation mechanism still needs to be further discussed. The study of S. zoogloeoides SHE10 provides scientific theoretical reference for expanding the resource pool of electroproducing bacteria and the types of electroproducing microorganisms.

Physical layer encryption scheme based on cellular automata and DNA encoding by hyper-chaos in a CO-OFDM system.

This study proposes an encryption scheme combining cellular automata (CA) and DNA encoding to improve security of a coherent optical orthogonal frequency division multiplexing (CO-OFDM) system, wherein key sequences are generated with good randomness and unpredictability by a 4-dimensional hyper-chaotic system. A base scrambling pseudo random binary sequence (PRBS) generated by the CA is introduced, which results in better scrambling effect and randomness in the conventional complex DNA encoding. The randomness, complexity and security of the system is enhanced due to 6 variable keys (key space of ∼10138). An experiment conducted in a 40 GHz 16QAM CO-OFDM system over an 80 km standard single mode fiber (SSMF) shows that the authorized user can successfully decrypt the received signal, while the eavesdroppers cannot derive useful information with bit error rate (BER) at approximately 0.5. An allowable optical signal to noise ratio (OSNR) penalty of 0.5 dB will be introduced to achieve same BER before and after encryption due to the error propagation of cellular automata.

Structure and Peptidome of the Bat MHC Class I Molecule Reveal a Novel Mechanism Leading to High-Affinity Peptide Binding.

Bats are natural reservoir hosts, harboring more than 100 viruses, some of which are lethal to humans. The asymptomatic coexistence with viruses is thought to be connected to the unique immune system of bats. MHC class I (MHC I) presentation is closely related to cytotoxic lymphocyte immunity, which plays an important role in viral resistance. To investigate the characteristics of MHC I presentation in bats, the crystal structures of peptide-MHC I complexes of Pteropus alecto, Ptal-N*01:01/HEV-1 (DFANTFLP) and Ptal-N*01:01/HEV-2 (DYINTNLVP), and two related mutants, Ptal-N*01:01/HEV-1PΩL (DFANTFLL) and Ptal-N*01:01ΔMDL/HEV-1, were determined. Through structural analysis, we found that Ptal-N*01:01 had a multi-Ala-assembled pocket B and a flexible hydrophobic pocket F, which could accommodate variable anchor residues and allow Ptal-N*01:01 to bind numerous peptides. Three sequential amino acids, Met, Asp, and Leu, absent from the α1 domain of the H chain in other mammals, were present in this domain in the bat. Upon deleting these amino acids and determining the structure in p/Ptal-N*01:01ΔMDL/HEV-1, we found they helped form an extra salt-bridge chain between the H chain and the N-terminal aspartic acid of the peptide. By introducing an MHC I random peptide library for de novo liquid chromatography-tandem mass spectrometry analysis, we found that this insertion module, present in all types of bats, can promote MHC I presentation of peptides with high affinity during the peptide exchange process. This study will help us better understand how bat MHC I presents high-affinity peptides from an extensive binding peptidome and provides a foundation to understand the cellular immunity of bats.

Mechanism of the Conformational Change of the Protein Methyltransferase SMYD3: A Molecular Dynamics Simulation Study.

SMYD3 is a SET-domain-containing methyltransferase that catalyzes the transfer of methyl groups onto lysine residues of substrate proteins. Methylation of MAP3K2 by SMYD3 has been implicated in Ras-driven tumorigenesis, which makes SMYD3 a potential target for cancer therapy. Of all SMYD family proteins, SMYD3 adopt a closed conformation in a crystal structure. Several studies have suggested that the conformational changes between the open and closed forms may regulate the catalytic activity of SMYD3. In this work, we carried out extensive molecular dynamics simulations on a series of complexes with a total of 21 µs sampling to investigate the conformational changes of SMYD3 and unveil the molecular mechanisms. Based on the C-terminal domain movements, the simulated models could be depicted in three different conformational states: the closed, intermediate and open states. Only in the case that both the methyl donor binding pocket and the target lysine-binding channel had bound species did the simulations show SMYD3 maintaining its conformation in the closed state, indicative of a synergetic effect of the cofactors and target lysine on regulating the conformational change of SMYD3. In addition, we performed analyses in terms of structure and energy to shed light on how the two regions might regulate the C-terminal domain movement. This mechanistic study provided insights into the relationship between the conformational change and the methyltransferase activity of SMYD3. The more complete understanding of the conformational dynamics developed here together with further work may lay a foundation for the rational drug design of SMYD3 inhibitors.

A potentially important resource: endophytic yeasts.

Recent advancements in the research on endophytes isolated from plants and crops have greatly broadened its application in various fields. Endophytic bacteria and endophytic fungi are known to promote the growth of various plants. Besides, the secondary metabolites such as alcohol and xylitol secreted by the endophytic yeast also help their hosts to resist microbial invasion. This makes them a potential substitute for chemical-based control methods. Moreover, the plant hosts can also provide nutrients for the growth of endophytic yeasts. To achieve the symbiotic relationship, yeasts must colonize most parts of the plant tissues, including intercellular spaces, cytoplasm, stomata of seeds, roots, stems, leaves, and fruits as well. Conventionally, isolation of endophytic yeasts from different plant tissues and understanding their interior plants colonization mechanism have remainedkey strategies to exploit their key potentials. In this review, we will elaborate on the diversity, characteristics of colonization, and the factors that influence the distribution of endophytic yeasts. This review also lays a theoretical foundation for the application of endophytic yeasts in various industrial and agricultural practices.

Distribution of ancient α1 and α2 domain lineages between two classical MHC class I genes and their alleles in grass carp.

Major histocompatibility complex (MHC) class I molecules play a crucial role in the immune response by binding and presenting pathogen-derived peptides to specific CD8+ T cells. From cDNA of 20 individuals of wild grass carp (Ctenopharyngodon idellus), we could amplify one or two alleles each of classical MHC class I genes Ctid-UAA and Ctid-UBA. In total, 27 and 22 unique alleles of Ctid-UAA and Ctid-UBA were found. The leader, α1, transmembrane and cytoplasmic regions distinguish between Ctid-UAA and Ctid-UBA, and their encoded α1 domain sequences belong to the ancient lineages α1-V and α1-II, respectively, which separated several hundred million years ago. However, Ctid-UAA and Ctid-UBA share allelic lineage variation in their α2 and α3 sequences, in a pattern suggestive of past interlocus recombination events that transferred α2+α3 fragments. The allelic Ctid-UAA and Ctid-UBA variation involves ancient variation between domain lineages α2-I and α2-II, which in the present study was dated back to before the ancestral separation of teleost fish and spotted gar (> 300 million years ago). This is the first report with compelling evidence that recombination events combining different ancient α1 and α2 domain lineages had a major impact on the allelic variation of two different classical MHC class I genes within the same species.

Major Histocompatibility Complex Class I (FLA-E*01801) Molecular Structure in Domestic Cats Demonstrates Species-Specific Characteristics in Presenting Viral Antigen Peptides.

Feline immunodeficiency virus (FIV) infection in domestic cats is the smallest usable natural model for lentiviral infection studies. FLA-E*01801 was applied to FIV AIDS vaccine research. We determined the crystal structure of FLA-E*01801 complexed with a peptide derived from FIV (gag positions 40 to 48; RMANVSTGR [RMA9]). The A pocket of the FLA-E*01801 complex plays a valuable restrictive role in peptide binding. Mutation experiments and circular-dichroism (CD) spectroscopy revealed that peptides with Asp at the first position (P1) could not bind to FLA-E*01801. The crystal structure and in vitro refolding of the mutant FLA-E*01801 complex demonstrated that Glu63 and Trp167 in the A pocket play important roles in restricting P1D. The B pocket of the FLA-E*01801 complex accommodates M/T/A/V/I/L/S residues, whereas the negatively charged F pocket prefers R/K residues. Based on the peptide binding motif, 125 FLA-E*01801-restricted FIV nonapeptides (San Diego isolate) were identified. Our results provide the structural basis for peptide presentation by the FLA-E*01801 molecule, especially A pocket restriction on peptide binding, and identify the potential cytotoxic T lymphocyte (CTL) epitope peptides of FIV presented by FLA-E*01801. These results will benefit both the reasonable design of FLA-E*01801-restricted CTL epitopes and the further development of the AIDS vaccine.IMPORTANCE Feline immunodeficiency virus (FIV) is a viral pathogen in cats, and this infection is the smallest usable natural model for lentivirus infection studies. To examine how FLA I presents FIV epitope peptides, we crystallized and solved the first classic feline major histocompatibility complex class I (MHC-I) molecular structure. Surprisingly, pocket A restricts peptide binding. Trp167 blocks the left side of pocket A, causing P1D to conflict with Glu63 We also identified the FLA-E*01801 binding motif X (except D)-(M/T/A/V/I/L/S)-X-X-X-X-X-X-(R/K) based on structural and biochemical experiments. We identified 125 FLA-E*01801-restricted nonapeptides from FIV. These results are valuable for developing peptide-based FIV and human immunodeficiency virus (HIV) vaccines and for studying how MHC-I molecules present peptides.

Computational complexity comparison of single-carrier DMT and conventional DMT in data center interconnect.

High peak-to-average power ratio (PAPR) is an inherent defect in intensity modulation and direct detection (IM/DD) discrete-multitone (DMT) system, which will cause serious signal nonlinear distortion over fiber transmission. Single carrier-DMT (SC-DMT), which also refers to the discrete-Fourier-transform spread DMT (DFT-spread DMT), is a promising technology for DMT signal PAPR reduction, but higher computational complexity is required due to the additional DFT/IDFT operations in transceiver. In this paper, we experimentally compare the performance of SC-DMT and conventional DMT (CDMT) signal when the computational complexity of SC-DMT transceiver is lower than CDMT by reducing the FFT size in SC-DMT. The results show that the receiver sensitivity of 20 GHz 1024-point FFT based SC-DMT improves by 0.7 dB than 8192-point FFT based CDMT for both 120 Gb/s 64QAM-DMT and 140 Gb/s 128QAM-DMT signal transmission over 2-km single mode fiber (SMF) at the BER of 3.8 × 10-3 and 2.0 × 10-2, respectively. It is the first time to find that the SC-DMT with lower transceiver computational complexity outperforms CDMT. In addition, fast-Hartley-transform (FHT) technique is employed to replace FFT for further transceiver computational complexity reduction. The results give out that FHT-based SC-DMT shows the same BER performance with FFT-based SC-DMT, while the computational complexity of the transceiver can be reduced by half.

Comparison of null-subcarriers reservation and adaptive notch filter for narrowband interference cancellation in intra-data center interconnect with DMT signal transmission.

In this paper, we experimentally compare the performance of two different narrowband interference suppression schemes in 120 Gb/s intensity modulation and direct detection (IM/DD) system with discrete multi-tone (DMT) signal transmission for intra-data center interconnect (Intra-DCI). Digital pre-equalization and DFT-spread techniques are applied for system bandwidth limitation induced signal distortion compensation and signal peak to average power ratio (PAPR) reduction, respectively. Null-subcarriers reservation (NSR) and adaptive notch filter (ANF) techniques are compared during the suppression of digital-to-analog convertor (DAC) clock leakage induced narrowband interference. 1.2 dB and 1.8 dB DMT receiver sensitivity improvements can be achieved at a bit-error rate of 3.8 × 10-3 in optical back-to-back (OBTB) transmission when optimized NSR and ANF schemes are applied for narrowband interference cancellation, respectively. After 2-km single mode fiber (SMF) transmission, the required received optical power (ROP) of DMT signal with optimized NSR and ANF for narrowband interference cancellation at BER of 3.8 × 10-3 are -6.5 dBm and -7.1 dBm, respectively. Obviously, ANF outperforms NSR scheme in narrowband interference cancellation for DMT system.

Optical I/Q modulation utilizing dual-drive MZM for fiber-wireless integration system at Ka-band.

In this Letter, a fiber-wireless integration system at Ka-band adopting heterodyne coherent detection is proposed and experimentally demonstrated. Optical I/Q up-conversion is realized with a low-cost dual-drive Mach-Zehnder modulator (DD-MZM) instead of the traditional I/Q nested MZM. To avoid non-convergence during constant-modulus-algorithm-based equalization, DC elimination is applied to suppress the ultra-high peak in the frequency domain after frequency down-conversion and symbol-phase-average-processing-based coarse phase noise estimation. Using DD-MZM for optical I/Q modulation, transmission, and reception of single polarization, 20-Gbit/s quadrature phase shift keying (QPSK) over 40-km single-mode fiber (SMF) and 5-m free space at Ka-band is demonstrated.

Identification of Novel Endophytic Yeast Strains from Tangerine Peel.

Seven endophytic yeast strains were isolated from tangerine peel (Citrus reticulata Blanco) and genotyped through clustering with D1/D2 and ITS1-5.8S-ITS2 sequences from GenBank. Phenotypic characteristics were obtained through commercial kits and through assisted species identification. Indole-3-acetic acid (IAA) production by the yeast strains was assessed using Salkowski reagent and High-Performance Liquid chromatography (HPLC). The growth-promoting effects of the yeast were evaluated using the 'ragdoll' method. CRYb1, CRYb2 and CRYb7 isolates were identified as the closest species Hanseniaspora opuntiae. CRYb3 was identified as Pichia kluyveri. CRYb4, CRYb5 and CRYb6 were identified as Meyerozyma guilliermondii. CRYb1, CRYb5, CRYb6 and CRYb7 were found to be capable of IAA production. The most promising yeast strains now require further evaluation for their ability to promote plant growth in vitro and in vivo. These data increase our knowledge of the distribution and biological properties of endophytic yeast. This is important information that will be required to fully harness the growth-promoting properties of yeast strains.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of swine leukocyte antigen 2 complexed with a CTL epitope AS64 derived from Asia1 serotype of foot-and-mouth disease virus.

BACKGROUND: Currently, the structural characteristics of the swine major histocompatibility complex (MHC) class I molecule, also named swine leukocyte antigen class I (SLA-I) molecule need to be further clarified. RESULTS: A complex of SLA-I constituted by an SLA-2*HB01 molecule with swine ß2-microglobulin and a cytotoxic T lymphocyte (CTL) epitope FMDV-AS64 (ALLRSATYY) derived from VP1 protein (residues 64-72) of Asia 1 serotype of foot-and-mouth disease virus (FMDV) was expressed, refolded, purified and crystallized. By preliminary X-ray diffraction analysis, it was shown that the diffraction resolution of the crystal was 2.4 Å and the space group belonged to P212121 with unit cell parameters a = 48.37, b = 97.75, c = 166.163 Å. CONCLUSION: This research will be in favor of illuminating the structural characteristics of an SLA-2 molecule associated with a CTL epitope derived from Asia1 serotype of FMDV.

TFEB coordinates autophagy and pyroptosis as hepatotoxicity responses to ZnO nanoparticles.

Zinc oxide nanoparticles (ZnO NPs) have drawn serious concerns about their biotoxicity due to their extensive applications in biological medicine, clinical therapeutic, daily chemical production, food and agricultural additives. In our present study, we clarified hepatotoxic mechanism of ZnO NPs through investigating the crosstalk between autophagy and pyroptosis, a remaining enigma in hepatocyte stimulated by ZnO NPs. Based on the effects of autophagy intervention by Rapamycin (Rap) and 3-Methyladenine (3-MA), and the observation of pyroptosis morphology and related indexes, the autophagy and pyroptosis simultaneously initiated by ZnO NPs were interrelated and the autophagy characterized by autophagosome production and increased expression of autophagy proteins was identified as a protective response of ZnO NPs against pyroptosis. According to the analysis of protein expression and fluorescence localization, the NLRP3 inflammasome assemble and the classical Caspase-1/GSDMD-dependent pyroptosis induced by ZnO NPs was modulated by autophagy. In this process, the adjustment of TFEB expression and nuclear translocation by gene knockout and gene overexpression, further altered the tendency of ZnO NPs-induced pyroptosis via the regulation of autophagy and lysosomal biogenesis. The knockout of TFEB gene exacerbated the pyroptosis via autophagy elimination and lysosome inhibition. While the alleviation of NLRP3 generation and pyroptosis activation was observed after treatment of TFEB gene overexpression. Additionally, the siRNA interference confirmed that TRAF-6 was involved in the TFEB-mediated global regulation of autophagy-lysosome-pyroptosis in response to ZnO NPs. Accordingly, pyroptosis induced by ZnO NPs in hepatocyte could be significantly avoided by TFEB-regulated autophagy and lysosome, further providing new insights for the risk assessment and therapeutic strategy.

The Parallel Presentation of Two Functional CTL Epitopes Derived from the O and Asia 1 Serotypes of Foot-and-Mouth Disease Virus and Swine SLA-2*HB01: Implications for Universal Vaccine Development.

Foot-and-mouth disease virus (FMDV) poses a significant threat to the livestock industry. Through their recognition of the conserved epitopes presented by the swine leukocyte antigen (SLA), T cells play a pivotal role in the antiviral immunity of pigs. Herein, based on the peptide binding motif of SLA-2*HB01, from an original SLA-2 allele, a series of functional T-cell epitopes derived from the dominant antigen VP1 of FMDV with high binding capacity to SLA-2 were identified. Two parallel peptides, Hu64 and As64, from the O and Asia I serotypes, respectively, were both crystallized with SLA-2*HB01. Compared to SLA-1 and SLA-3, the SLA-2 structures showed the flexibility of residues in the P4, P6, and P8 positions and in their potential interface with TCR. Notably, the peptides Hu64 and As64 adopted quite similar overall conformation when bound to SLA-2*HB01. Hu64 has two different conformations, a more stable 'chair' conformation and an unstable 'boat' conformation observed in the two molecules of one asymmetric unit, whereas only a single 'chair' conformation was observed for As64. Both Hu64 and As64 could induce similar dominant T-cell activities. Our interdisciplinary study establishes a basis for the in-depth interpretation of the peptide presentation of SLA-I, which can be used toward the development of universal vaccines.

The unique structure of the zebrafish TNF-α homotrimer.

In the current study, zebrafish TNF-α1 (zTNF-α1) was crystallized, and the structure was analyzed. The zTNF-α1 trimer is composed of three monomers whose height and width are 50 Å and 60 Å, respectively. Compared with human TNF-α, zTNF-α1 shows only ~30% amino acid identity, the EF loop of each monomer lacks three amino acids, the CD loop is increased by four amino acids, and the AA'' loop is increased by one amino acid. In addition, an A″-ß-chain is added to the zTNF-α1 monomer, forming two ß-sheet layers with 6:5 ß-chains. The top of the trimer is missing three amino acids and the inner coil because the EF loop seals the central hole at the top, forming a unique structure. In conclusion, the results elucidated the structure of the zTNF-α1 trimer, providing immunological knowledge for studying TNF-α function in the zebrafish animal model and structural information for exploring TNF-α family evolution.

Peptidomes and Structures Illustrate Two Distinguishing Mechanisms of Alternating the Peptide Plasticity Caused by Swine MHC Class I Micropolymorphism.

The micropolymorphism of major histocompatibility complex class I (MHC-I) can greatly alter the plasticity of peptide presentation, but elucidating the underlying mechanism remains a challenge. Here we investigated the impact of the micropolymorphism on peptide presentation of swine MHC-I (termed swine leukocyte antigen class I, SLA-I) molecules via immunopeptidomes that were determined by our newly developed random peptide library combined with the mass spectrometry (MS) de novo sequencing method (termed RPLD-MS) and the corresponding crystal structures. The immunopeptidomes of SLA-1*04:01, SLA-1*13:01, and their mutants showed that mutations of residues 156 and 99 could expand and narrow the ranges of peptides presented by SLA-I molecules, respectively. R156A mutation of SLA-1*04:01 altered the charge properties and enlarged the volume size of pocket D, which eliminated the harsh restriction to accommodate the third (P3) anchor residue of the peptide and expanded the peptide binding scope. Compared with 99Tyr of SLA-1*0401, 99Phe of SLA-1*13:01 could not form a conservative hydrogen bond with the backbone of the P3 residues, leading to fewer changes in the pocket properties but a significant decrease in quantitative of immunopeptidomes. This absent force could be compensated by the salt bridge formed by P1-E and 170Arg. These data illustrate two distinguishing manners that show how micropolymorphism alters the peptide-binding plasticity of SLA-I alleles, verifying the sensitivity and accuracy of the RPLD-MS method for determining the peptide binding characteristics of MHC-I in vitro and helping to more accurately predict and identify MHC-I restricted epitopes.

The Mechanism of ß2m Molecule-Induced Changes in the Peptide Presentation Profile in a Bony Fish.

Contemporary antigen presentation knowledge is based on the existence of a single ß2m locus, and a classical MHC class I forms a complex with a peptide (i.e., pMHC-I) to trigger CTL immunity. However, two ß2m loci have been found in diploid bony fish; the function of the two ß2m molecules is unclear. Here, we determined the variant peptide profiles originating from different products of the ß2m loci binding to the same MHC-I molecule and further solved the crystal structures of the two pMHC-I molecules (i.e., pCtid-UAA-ß2m-2 and pCtid-UAA-ß2m-1-II). Of note, in pCtid-UAA-ß2m-2, a unique hydrogen bond network formed in the bottom of the peptide-binding groove (PBG) led to α2-helix drift, ultimately leading to structural changes in the PBG. The mechanism of the change in peptide presentation profiles by ß2m molecules is illustrated. The results are also of great significance for antivirus and antitumor functions in cold-blooded vertebrates and even humans.