Whey Acidic Protein Research Articles

223 The novel whey acidic protein WFDC12 is expressed in particular terminally differentiated keratinocytes

223 The novel whey acidic protein WFDC12 is expressed in particular terminally differentiated keratinocytes

The expression and clinical significance of secretory leukocyte proteinase inhibitor (SLPI) in mammary carcinoma using bioinformatics analysis

BackgroundSecretory leukocyte protease inhibitor (SPLI) was a secreted protein which belongs to a member of whey acidic protein four-disulfide core family. In breast cancer (BC) it may inhibit cell proliferation and promote cancer metastasis. In this study, a comprehensive bioinformatics analysis was performed to identify the expression and prognostic value of SLPI in breast cancer. MethodsSLPI expression in breast cancer was analyzed in Oncomine online database, which was subsequently confirmed by quantitative PCR (qPCR) in 18 BC samples and western blotting in 26 BC samples. Breast cancer gene-expression miner v4.1 was used to access the expression level with clinicopathological parameters in breast cancer patients. The prognostic values of SLPI in breast cancer were evaluated using the PrognoScan database. ResultsOur results indicated that SLPI was downregulated in breast cancer than in normal tissues. SLPI expression was found to be negatively correlated with estrogen receptor (ER) and progesterone receptor (PR) status. SLPI expression level was decreased in negative basal-like status patients compared with positive basal-like status. Meanwhile, triple-negative breast cancer status positive correlated with SLPI. We confirmed a positive correlation between SLPI and interleukin 17 receptor B (IL17RB) express in breast cancer tissues via oncomine co-expression analysis. Ten proteins: Elastase, Granulin, Lipocalin, Defensin beta 103B, Defensin beta 103A, Tubulin, Heparin-binding EGF-like growth factor, Interleukin 6, Epidermal growth factor, Phospholipid scramblase 1 were determinate interactions with SLPI by STRING. ConclusionSLPI could as a biomarker to predict the prognosis values of breast cancer. However, further comprehensive study and mining more evidence are needed to clarify our results.

Use of acid whey protein concentrate as an ingredient in nonfat cup set-style yogurt

Acid whey resulting from the production of soft cheeses is a disposal problem for the dairy industry. Few uses have been found for acid whey because of its high ash content, low pH, and high organic acid content. The objective of this study was to explore the potential of recovery of whey protein from cottage cheese acid whey for use in yogurt. Cottage cheese acid whey and Cheddar cheese whey were produced from standard cottage cheese and Cheddar cheese-making procedures, respectively. The whey was separated and pasteurized by high temperature, short time pasteurization and stored at 4°C. Food-grade ammonium hydroxide was used to neutralize the acid whey to a pH of 6.4. The whey was heated to 50°C and concentrated using ultrafiltration and diafiltration with 11 polyethersulfone cartridge membrane filters (10,000-kDa cutoff) to 25% total solids and 80% protein. Skim milk was concentrated to 6% total protein. Nonfat, unflavored set-style yogurts (6.0 ± 0.1% protein, 15 ± 1.0% solids) were made from skim milk with added acid whey protein concentrate, skim milk with added sweet whey protein concentrate, or skim milk concentrate. Yogurt mixes were standardized to lactose and fat of 6.50% and 0.10%, respectively. Yogurt was fermented at 43°C to pH 4.6 and stored at 4°C. The experiment was replicated in triplicate. Titratable acidity, pH, whey separation, color, and gel strength were measured weekly in yogurts through 8 wk. Trained panel profiling was conducted on 0, 14, 28, and 56 d. Fat-free yogurts produced with added neutralized fresh liquid acid whey protein concentrate had flavor attributes similar those with added fresh liquid sweet whey protein but had lower gel strength attributes, which translated to differences in trained panel texture attributes and lower consumer liking scores for fat-free yogurt made with added acid whey protein ingredient. Difference in pH was the main contributor to texture differences, as higher pH in acid whey protein yogurts changed gel structure formation and water-holding capacity of the yogurt gel. In a second part of the study, the yogurt mix was reformulated to address texture differences. The reformulated yogurt mix at 2% milkfat and using a lower level of sweet and acid whey ingredient performed at parity with control yogurts in consumer sensory trials. Fresh liquid acid whey protein concentrates from cottage cheese manufacture can be used as a liquid protein ingredient source for manufacture of yogurt in the same factory.

2233-PUB: Clinical Features and Biomarkers of Nonalbuminuric and Albuminuric Patterns of Chronic Kidney Disease in Subjects with Type 2 Diabetes

The Aim: to define the phenotypes of non-albuminuric and albuminuric chronic kidney disease (NA-CKD and A-CKD) in subjects with type 2 diabetes (T2D). Methods: One hundred and eighty four T2D patients were included in the study. Patients with urinary albumin to creatinine ratio (UACR) <3 mg/mmol and estimated glomerular filtration rate (eGFR) <60 ml/min×1.73 m2 were classified as NA-CKD group (n=111). Individuals with persistent UACR >3 mg/mmol and eGFR <60 ml/min×1.73 m2 were considered as A-CKD patients (n=73). Urinary nephrin and podocin, as podocyte injury markers, and whey acidic protein four-disulfide core domain protein 2 (WFDC-2), which is considered as a marker of tubulointerstitial involvement, were assessed by ELISA and compared to control (20 nondiabetic subjects). Results: The NA-CKD pattern was associated with female sex (p=0.04), reduced HbA1c levels (p=0.04) and diuretic use (p=0.001). Patients with A-CKD, as compared to NA-CKD one, demonstrated lower body weight (p=0.02), waist-to-hip ratio (p=0.009), HDL-cholesterol (p=0.01), and lymphocyte count (p=0.002). Age, diabetes duration, the prevalence of diabetic retinopathy, coronary artery disease, myocardial infarction, chronic heart failure and stroke did not differ between the groups. The nephrin and podocin concentrations, adjusted to urinary creatinine, were increased significantly in subjects with A-CKD only (p=0.002 and 0.04 respectively). The excretion of WFDC-2 was elevated in both NA-CKD and A-CKD groups (p=0.01 and p=0.0003 respectively). Conclusion: In T2D subjects, female gender, acceptable glycemic control and treatment with diuretics are associated with NA-CKD, whereas abdominal obesity, poor glycemic control, and elevated urinary excretion of podocyte markers are the features of A-CKD. Urinary WFDC-2 excretion is increased in both CKD phenotypes. Disclosure A.I. Korbut: None. V. Klimontov: Advisory Panel; Self; Sanofi. Speaker's Bureau; Self; AstraZeneca, Boehringer Ingelheim International GmbH, Sanofi.

Lack of whey acidic protein (WAP) four-disulfide core domain protease inhibitor 2 (WFDC2) causes neonatal death from respiratory failure in mice.

ABSTRACTRespiratory failure is a life-threatening problem for pre-term and term infants, yet many causes remain unknown. Here, we present evidence that whey acidic protein (WAP) four-disulfide core domain protease inhibitor 2 (Wfdc2), a protease inhibitor previously unrecognized in respiratory disease, may be a causal factor in infant respiratory failure. Wfdc2 transcripts are detected in the embryonic lung and analysis of a Wfdc2-GFP knock-in mouse line shows that both basal and club cells, and type II alveolar epithelial cells (AECIIs), express Wfdc2 neonatally. Wfdc2-null-mutant mice display progressive atelectasis after birth with a lethal phenotype. Mutant lungs have multiple defects, including impaired cilia and the absence of mature club cells from the tracheo-bronchial airways, and malformed lamellar bodies in AECIIs. RNA sequencing shows significant activation of a pro-inflammatory pathway, but with low-quantity infiltration of mononuclear cells in the lung. These data demonstrate that Wfdc2 function is vitally important for lung aeration at birth and that gene deficiency likely causes failure of the lung mucosal barrier.

Anti-Vibrio Response of CarcininPm 1 from Penaeus monodon and Its Heterologous Expression

Crustins are crucial antimicrobial peptides in shrimp and play very important roles in innate immunity. In this research, a Type I crustin from Penaeus monodon (CarcininPm1) contained 108 residues was studied. The first 16 residues are signal peptide. It contained ten cysteines but did not form an intact whey acidic protein (WAP) domain. CarcininPm1 was observed to widely distribute in all tissues, while highly expressed in intestine. The expression level of CarcininPm1 in hepatopancreas was up-regulated 12- 20 times during 4-12h post challenged by Vibrio parahaemolyticus. And the transcription in heart, stomach and gills was also significantly enhanced at 4h post challenge. The mature peptide was expressed successfully in Eschericha coli by fusing to a SUMO protein, with protein production around 8 mg/mL. After cleavage with SUMO protease, carcininPm1 was obtained indicating its potential applications.

HcCUB-Lec, a newly identified C-type lectin that contains a distinct CUB domain and participates in the immune defense of the triangle sail mussel Hyriopsis cumingii

As pattern recognition receptors (PRRs), C-type lectins (CTLs) play crucial roles in recognizing and eliminating pathogens in innate immunity. In this study, a novel CTL (HcCUB-Lec) was identified from the triangle sail mussel Hyriopsis cumingii. The full-length of HcCUB-Lec cDNA was 1558 bp with an open reading frame of 1281 bp that encodes a putative protein of 426 amino acid residues, including an N-terminal signal peptide, a complement Uegf Bmp1 (CUB) domain, a single carbohydrate recognition domain (CRD), and a transmembrane domain. Quantitative real-time PCR analysis revealed that HcCUB-Lec transcript was distributed in all examined tissues with the highest levels in hepatopancreas and was significantly upregulated in gills and hepatopancreas after immune challenge with Staphyloccocus aureus and Vibrio parahaemolyticus. When HcCUB-Lec was silenced by RNAi, the expression levels of three antimicrobial peptides, including whey acidic protein (HcWAP), defensin (HcDef), and lysozyme (HcLyso), were dramatically decreased in gills. The recombinant HcCUB-Lec and its individual CUB and CRD domains can bind with Gram-positive bacteria (S. aureus and Bacillus subtilis), Gram-negative bacteria (V. parahaemolyticus and Aeromonas hydrophila), and polysaccharides (lipopolysaccharide and peptidoglycan). Moreover, rHcCUB-Lec and its domains could also agglutinate S. aureus and V. parahaemolyticus in the presence of Ca2+ and can clear V. parahaemolyticus in H. cumingii. Results of this study suggest that HcCUB-Lec acts as an antimicrobial PRR that participates in the innate immune responses of H. cumingii.

Molecular cloning and functional characterization of a short peptidoglycan recognition protein from triangle-shell pearl mussel (Hyriopsis cumingii)

Peptidoglycan (PGN) is an important target of recognition in invertebrate innate immunity. PGN recognition proteins (PGRPs) are responsible for PGN recognition. In this study, we cloned and functionally analyzed a short PGRP (HcPGRP2) from the triangle-shell pearl mussel Hyriopsis cumingii. The full-length cDNA sequence of HcPGRP2 gene was 1185 bp containing an open reading frame of 882 bp encoding a 293 amino acid protein. HcPGRP2 was predicted to have two SH3b domains and a conserved C-terminal PGRP domain. Quantitative real-time RT-PCR showed that HcPGRP2 was expressed in all examined tissues and its expression was induced most significantly by Staphylococcus aureus and Vibrio parahaemolyticus in the hepatopancreas and gills. RNA interference by siRNA results revealed that HcPGRP2 was involved in the regulation of whey acidic protein, theromacin, and defensin expression. As a pattern-recognition receptor, recombinant HcPGRP2 (rHcPGRP2) protein can bind and agglutinate (Ca2+ dependent) all tested bacteria. rHcPGRP2 exhibited specific binding to PGN but not to lipopolysaccharide. Moreover, rHcPGRP2 inhibited the growth activities of S. aureus and V. parahaemolyticus in vitro and accelerated the clearance of V. parahaemolyticus in vivo. Overall, our results indicated that HcPGRP2 may play an important role in the antibacterial immune mechanisms of H. cumingii.

Acid whey proteolysis to produce angiotensin-I converting enzyme inhibitory hydrolyzate

α-Lactalbumin (ALA) and β-lactoglobulin (BLG) are the main proteins in acid whey and proteolytic digestion of these proteins provides a source of peptides that inhibit angiotensin-I converting enzyme (ACE-I). Enzymatic hydrolysis of acid whey proteins using cysteine peptidase enzyme extract (antiacanthain) from ripe Bromelia antiacantha Bertol fruit was investigated. The effect of pH on hydrolysis kinetics of ALA and BLG by antiacanthain was analysed at 50 °C. Antiacanthain had a high catalytic efficiency for ALA (1.3 × 104 M−1 s−1) and BLG (1.7 × 104 M−1 s−1) at pH 9.2. The effect of temperature and amount of antiancanthain (enzyme to substrate ratio or E/S) on acid whey hydrolysis were studied by measurement of degree of hydrolysis (DH) and protein degradation profile evaluated by tricine–sodium dodecyl sulfate-polyacrylamide gel electrophoresis (tricine–SDS-PAGE). Optimal acid whey hydrolysis was at pH 9.2, E/S = 1.0 (U/mg) and 50 °C. BLG was degraded faster than that of ALA, but after 30 min hydrolysis almost all BLG and ALA were degraded and new bands corresponding to peptides with a mass lower than 3.0 kDa appeared. Fractions of acid whey (DH = 12.3%), ALA (DH = 17.3%) and BLG (DH = 30.6%) hydrolyzates showed ACE-I inhibitory activity. Results also suggest that most inhibitory activity might be due to low molecular weight peptides from ALA, since 3 kDa permeate of ALA hydrolyzate showed the highest IC50 value (1.7 ± 0.4 µg/mL). Interestingly, low molecular weight peptides from both ALA and BLG had low immunoreactivity levels, monitored by latex agglutination tests, suggesting that these peptides could be safe for use by those with strong allergic responses. Results indicate that antiacanthain can be used as an enzymatic preparation for acid whey hydrolysis. The potential use of this hydrolyzate for manufacture of health-promoting products of high added value is discussed.

Trichuris muris whey acidic protein induces type 2 protective immunity against whipworm.

Human whipworm (Trichuris trichiura) infects approximately 1 in 15 people worldwide, representing the leading infectious cause of colitis and subsequent, inflammatory bowel disease (IBD). Current control measures focused on mass deworming have had limited success due to low drug efficacies. Vaccination would be an ideal, cost-effective strategy to induce protective immunity, leading to control of infection and transmission. Here we report the identification of whey acidic protein, a whipworm secretory protein, as a strong immunogen for inducing protective efficacy in a surrogate mouse T. muris infection model. The recombinant WAP protein (rTm-WAP49), as well as a single, highly conserved repeat within WAP (fragment 8) expressed as an Na-GST-1 fusion protein (rTm-WAP-F8+Na-GST-1), generate a strong T helper type 2 (Th2) immune response when delivered as subcutaneous vaccines formulated with Montanide ISA 720. Oral challenge with T. muris infective eggs following vaccination led to a significant reduction in worm burden of 48% by rTm-WAP49 and 33% by rTm-WAP-F8+Na-GST-1. The cellular immune correlates of protection included significant antigen-specific production of Th2 cytokines IL-4, IL-9, and IL-13 by cells isolated from the vaccine-draining inguinal lymph nodes, parasite-draining mesenteric lymph nodes, and spleen in mice vaccinated with either rTm-WAP49 or rTm-WAP-F8+Na-GST-1. The humoral immune correlates included a high antigen-specific ratio of IgG1 to IgG2a, without eliciting an IgE-mediated allergic response. Immunofluorescent staining of adult T. muris with WAP antisera identified the worm’s pathogenic stichosome organ as the site of secretion of native Tm-WAP protein into the colonic mucosa. Given the high sequence conservation for the WAP proteins from T. muris and T. trichiura, the results presented here support the WAP protein to be further evaluated as a potential human whipworm vaccine candidate.

Expression of the Shrimp wap gene in Drosophila elicits defense responses and protease inhibitory activity

The wap gene encodes a single whey acidic protein (WAP) domain-containing peptide from Chinese white shrimp (Fenneropenaeus chinensis), which shows broad-spectrum antimicrobial activities and proteinase inhibitory activities in vitro. To explore the medical applications of the WAP peptide, a wap gene transgenic Drosophila melanogaster was constructed. In wap-expressing flies, high expression levels of wap gene (>100 times) were achieved, in contrast to those of control flies, by qRT-PCR analysis. The wap gene expression was associated with increased resistance to microbial infection and decreased bacterial numbers in the flies. In addition, the WAP protein extract from wap-expressing flies, compared with control protein extract from control flies, showed improved antimicrobial activities against broad Gram-positive and Gram-negative bacteria, including the clinical drug resistant bacterium of methicillin-resistant S. aureus (MRSA), improved protease inhibitor activities against crude proteinases and commercial proteinases, including elastase, subtilis proteinase A, and proteinase K in vitro, and improved growth rate and microbial resistance, as well as wound-healing in loach and mouse models. These results suggest that wap-expressing flies could be used as a food additive in aquaculture to prevent infections and a potential antibacterial for fighting drug-resistant bacteria.

Comparative sequence analysis of crustin isoform MjCRS7 and MjWFDC-like gene from kuruma shrimp Marsupenaeus japonicus shows variant of the WFDC domain

Crustins are well known cysteine-rich cationic antimicrobial peptides (AMPs) in crustaceans that have WFDC [WAP (whey acidic protein) four-disulfide core] domain at the carboxyl terminus. Proteins containing a WFDC domain have been discovered in many invertebrates and vertebrates. Although, there have been many WFDC domain containing nucleotide sequences found in NCBI GenBank database, their distinct sequential characteristics and their role in the innate immune system is not well understood. Here, we identified a new crustin isoform from Marsupenaeus japonicus by transcriptome analysis. The full-length cDNA of this isoform (MjCRS7) consists of 537 bp that include a 489 bp open reading frame (ORF) encoding 162 deduced amino acids (aa). The sequence contains the eight conserved cysteine residues characteristic of the WFDC domain. A phylogenetic analysis showed that MjCRS7 is a type II crustin. We also identified the full-length cDNA of a M. japonicus MjWFDC-like gene. MjWFDC-like has a 543 bp ORF encoding 180 aa. In an RT-PCR analysis, MjCRS7 and MjWFDC-like transcripts were mainly detected in gill tissue. An alignment of MjCRS7 and MjWFDC-like with previously reported M. japonicus crustin isoform 1–5 (MjCRS1–5) showed variation in the WFDC-like domain. Neither of the genes was responsive to Vibrio parahaemolyticus, Vibrio penaeicida or white spot syndrome virus (WSSV) either by immersion or injection challenge test. Although crustins are mainly antimicrobial peptides, the present results suggest that MjCRS7 may have other roles in M. japonicus.

Identification of a 5‑lncRNA signature‑based risk scoring system for survival prediction in colorectal cancer.

The present study aimed to investigate potential prognostic long noncoding RNAs (lncRNAs) associated with colorectal cancer (CRC). An mRNA-seq dataset obtained from The Cancer Genome Atlas was employed to identify the differentially expressed lncRNAs (DELs) between CRC patients with good and poor prognoses. Subsequently, univariate and multivariate Cox regression analyses were conducted to analyze the prognosis-associated lncRNAs among all DELs. In addition, a risk scoring system was developed according to the expression levels of the prognostic lncRNAs, which was then applied to a training set and an independent testing set. Furthermore, the co-expressed genes of prognostic lncRNAs were screened using a Multi-Experiment Matrix online tool for construction of lncRNA-gene networks. Finally, Kyoto Encyclopedia of Genes and Genomes pathway and Gene Ontology (GO) function enrichment analyses were performed on genes in the lncRNA-gene networks using KOBAS, GOATOOLS and ClusterProfiler. The present study identified 82 DELs, of which long intergenic nonprotein coding RNA 2159, RP11-452L6.6, RP11-894P9.1 and RP11-69M1.6, and whey acidic protein four-disulfide core domain 21 (WFDC21P) were reported to be independently associated with the prognosis of patients with CRC. A 5-lncRNA signature-based risk scoring system was developed, which may be used to classify patients into low- and high-risk groups with significantly different recurrence-free survival times in the training and testing sets (P<0.05). Co-expressed genes of WFDC21P or RP11-69M1.6 were utilized to construct the lncRNA-gene networks. Genes in the networks were significantly enriched in ‘tight junction’, ‘focal adhesion’ and ‘regulation of actin cytoskeleton’ pathways, and numerous GO terms associated with ‘reactive oxygen species metabolism’ and ‘nitric oxide metabolism’. The present study proposed a 5-lncRNA signature-based risk scoring system for predicting the prognosis of patients with CRC, and revealed the associated signaling pathways and biological processes. The results of the present study may help improve prognostic evaluation in clinical practice.

683 The role of the WFDC [WAP (Whey acidic protein) four-disulfide core] family members in the epidermis

683 The role of the WFDC [WAP (Whey acidic protein) four-disulfide core] family members in the epidermis

Metabolomics Reveals Aryl Hydrocarbon Receptor Activation Induces Liver and Mammary Gland Metabolic Dysfunction in Lactating Mice.

The liver and the mammary gland have complementary metabolic roles during lactation. Substrates synthesized by the liver are released into the circulation and are taken up by the mammary gland for milk production. The aryl hydrocarbon receptor (AHR) has been identified as a lactation regulator in mice, and its activation has been associated with myriad morphological, molecular, and functional defects such as stunted gland development, decreased milk production, and changes in gene expression. In this study, we identified adverse metabolic changes in the lactation network (mammary, liver, and serum) associated with AHR activation using 1H nuclear magnetic resonance (NMR)-based metabolomics. Pregnant mice expressing Ahrd (low affinity) or Ahrb (high affinity) were fed diets containing beta naphthoflavone (BNF), a potent AHR agonist. Mammary, serum, and liver metabolomics analysis identified significant changes in lipid and TCA cycle intermediates in the Ahrb mice. We observed decreased amino acid and glucose levels in the mammary gland extracts of Ahrb mice fed BNF. The serum of BNF fed Ahrb mice had significant changes in LDL/VLDL (increased) and HDL, PC, and GPC (decreased). Quantitative PCR analysis revealed ∼50% reduction in the expression of key lactogenesis mammary genes including whey acid protein, α-lactalbumin, and β-casein. We also observed morphologic and developmental disruptions in the mammary gland that are consistent with previous reports. Our observations support that AHR activity contributes to metabolism regulation in the lactation network.

Cloning and Analysis of Gene Expression of Two Toll Receptors in Freshwater Pearl Mussel Hyriopsis cumingii

Toll receptors are involved in innate immunity of invertebrates. In this study, we identify and characterize two Toll genes (named HcToll4 and HcToll5) from triangle sail mussel Hyriopsis cumingii. HcToll4 has complete cDNA sequence of 3,162 bp and encodes a protein of 909 amino acids. HcToll5 cDNA is 4,501 bp in length and encodes a protein of 924 amino acids. Both deduced HcToll4 and HcToll5 protein contain signal peptide, extracellular leucine rich repeats (LRRs), and intracellular Toll/interleukin-1 (IL-1) receptor domains. Quantitative real-time PCR analysis revealed that HcToll4 and HcToll5 were largely distributed in the hepatopancreas and could be detected in the gills and mantle. HcToll4 and HcToll5 expression could respond to Staphylococcus aureus, Vibrio parahaemolyticus, White spot syndrome virus (WSSV) or Poly I:C challenge. RNA interference by siRNA results showed that HcToll4 and HcToll5 were involved in the regulation of theromacin (HcThe) and whey acidic protein (HcWAP) expression. Based on these results, HcToll4 and HcToll5 might play pivotal function in H. cumingii innate immune response.

Newly identified type II crustin (SpCrus2) in Scylla paramamosain contains a distinct cysteine distribution pattern exhibiting broad antimicrobial activity

Type II crustins are the most abundant type of crustins in shrimps that exhibit remarkable sequence diversities and broad antibacterial activities. This study characterized a novel type II crustin, SpCrus2, in the mud crab Scylla paramamosain. The SpCrus2 cDNA sequence is 620-bp long with a 495-bp open reading frame encoding a 164-amino acid protein. In the deduced protein, a 17-amino acid signal peptide, a glycine-rich hydrophobic region (GRR), and a cysteine-rich region (CRR) containing a whey acidic protein domain were predicted. SpCrus2 shares high similarity with most type II crustins (types IIa and IIb crustins) in shrimps but has a novel distribution pattern of cysteine residues that is distinct from most crustins. SpCrus2 and PlCrus3 from Pacifastacus leniusculus share high similarity and the same distribution pattern of cysteine residues. Thus, we proposed them as type IIc crustins. SpCrus2 is mainly distributed in the gills and can be up-regulated through Vibrio parahemolyticus or Staphylococcus aureus challenge. To investigate the biological functions of SpCrus2 and the underlying mechanisms, SpCrus2, GRR, CRR, and the mutant of CRR (CRR-M, the cysteine distribution pattern is mutated into that in most conventional crustins) were all overexpressed and purified. SpCrus2 GRR itself, as a glycine-rich amphiphilic peptide, exhibited evident antibacterial ability against Gram-negative bacteria, whereas CRR possessed potent antibacterial activity against Gram-positive bacteria. Either GRR or CRR exhibited weaker antibacterial activity than the whole protein of SpCrus2, indicating that GRR and CRR synergized to exert their potential antibacterial functions. In addition, CRR exhibited slightly stronger antimicrobial activity than CRR-M, suggesting that SpCrus2 containing this novel cysteine distribution pattern may exhibit stronger antimicrobial activity than most type II crustins with the conventional distribution pattern of cysteine residues. The likely antimicrobial ability of SpCrus2 may result from its microbial polysaccharide-binding and agglutination activities. Overall, this study characterized the first type II crustin in crabs and provided new insights into understanding the sequence and functional diversity of crustins and their immune functions in crustaceans.

Specific Molecular Signatures for Type II Crustins in Penaeid Shrimp Uncovered by the Identification of Crustin-Like Antimicrobial Peptides in Litopenaeus vannamei.

Crustins form a large family of antimicrobial peptides (AMPs) in crustaceans composed of four sub-groups (Types I-IV). Type II crustins (Type IIa or “Crustins” and Type IIb or “Crustin-like”) possess a typical hydrophobic N-terminal region and are by far the most representative sub-group found in penaeid shrimp. To gain insight into the molecular diversity of Type II crustins in penaeids, we identified and characterized a Type IIb crustin in Litopenaeus vannamei (Crustin-like Lv) and compared Type II crustins at both molecular and transcriptional levels. Although L. vannamei Type II crustins (Crustin Lv and Crustin-like Lv) are encoded by separate genes, they showed a similar tissue distribution (hemocytes and gills) and transcriptional response to the shrimp pathogens Vibrio harveyi and White spot syndrome virus (WSSV). As Crustin Lv, Crustin-like Lv transcripts were found to be present early in development, suggesting a maternal contribution to shrimp progeny. Altogether, our in silico and transcriptional data allowed to conclude that (1) each sub-type displays a specific amino acid signature at the C-terminal end holding both the cysteine-rich region and the whey acidic protein (WAP) domain, and that (2) shrimp Type II crustins evolved from a common ancestral gene that conserved a similar pattern of transcriptional regulation.

Construction of a universal recombinant expression vector that regulates the expression of human lysozyme in milk

The mammary gland provides a novel method for producing recombinant proteins in milk of transgenic animals. A key component in the technology is the construction of an efficient milk expression vector. Here, we established a simple method to construct a milk expression vector, by a combination of homologous recombination and digestion-ligation. Our methodology is expected to have the advantages of both plasmid and bacterial artificial chromosome (BAC) vectors. The BAC of mouse whey acidic protein gene (<i>mWAP</i>) was modified twice by homologous recombination to produce a universal expression vector, and the human lysozyme gene (<i>hLZ</i>) was then inserted into the vector by a digestion-ligation method. The final vector containing the 8.5 kb <i>mWAP</i> 5′ promoter, 4.8 kb <i>hLZ</i> genomic DNA, and 8.0 kb <i>mWAP</i> 3′ genomic DNA was microinjected into pronuclei of fertilized mouse embryos, to successfully generate two transgenic mouse lines that expressed recombinant human lysozyme (rhLZ) in milk. The highest expression level of rhLZ was 0.45 g·L⁻¹, and rhLZ exhibited the same antibacterial activity as native hLZ. Our results have provided a simple approach to construct a universal milk expression vector, and demonstrated that the resulting vector regulates the expression of hLZ in milk.

A single WAP domain (SWD)-containing protein with antiviral activity from Pacific white shrimp Litopenaeus vannamei

The single whey acidic protein (WAP) domain (SWD)-containing proteins, also called type III crustins, are a group of antimicrobial peptides (AMPs) in crustaceans. At present, a number of SWDs have been identified in shrimp, which showed essential antibacterial activities. However, the roles of SWDs in antiviral immune responses have not been reported up to now. In this study, a novel SWD (LvSWD3) was identified from Pacific white shrimp, Litopenaeus vannamei, which contained a typical single WAP domain homologous to those of other crustacean SWDs. Although lacking the pro and arg-rich region between the signal peptide and the WAP domain, LvSWD3 was closely clustered with other shrimp SWDs in the phylogenetic tree. Similar to many shrimp SWDs, the highest expression of LvSWD3 was detected in hemocytes. The LvSWD3 expression exhibited only limited changes after challenges with Vibrio parahaemolyticus, Poly (I:C) and lipopolysaccharide, but was significantly up-regulated after white spot syndrome virus (WSSV) infection. Silencing of LvSWDs significantly accelerated the death of the WSSV-infected but not the V. parahaemolyticus-infected shrimp. The recombinant LvSWD3 protein did not show proteinase inhibitory and antibacterial activities but could significantly postpone the death of WSSV-infected shrimp and reduce the viral load in tissues. These suggested that LvSWD3 was a novel SWD with antiviral activity.