Characterization of Multiple Alginate Lyases in a Highly Efficient Alginate-Degrading Vibrio Strain and Its Degradation Strategy.

Alginate is an important polysaccharide in the ocean that supports the growth of marine microorganisms. Many widespread Vibrio species possess alginate lyases and can utilize alginate as a carbon source, but the detailed alginate degradation mechanism in Vibrio remains to be further explored. In this study, we obtained a highly efficient alginate-degrading strain, Vibrio pelagius WXL662, with 11 alginate lyases (VpAly-I to -XI) and further elucidated its molecular mechanism of alginate degradation. Three alginate utilization loci (AUL) were identified in different parts of WXL662's genome, comprising six alginate lyases (VpAly-I, -II, -VIII, -IX, -X, and -XI) and other genes related to alginate degradation. Most of the alginate-degrading genes are strongly induced when alginate is provided as the sole carbon source. Ten alginate lyases (VpAly-I to -X) had been purified and characterized, including six from polysaccharide lyase family 7 (PL7), three from PL17, and one from PL6. These recombinant alginate lyases existing in different cellular locations were active at a wide temperature (10 to 50°C) and pH (4.0 to 9.0) range, with different substrate preferences and diverse degradation products, enabling WXL662 to efficiently utilize alginate in a changing marine environment. Importantly, outer membrane vesicles (OMVs) can act as vectors for alginate lyases (VpAly-II, -V, and -VI) in WXL662. Further investigations of public Vibrio genomes revealed that most alginate-degrading vibrios possess one AUL instead of previously reported "scattered" system. These results emphasize the specific alginate degradation strategy in Vibrio pelagius WXL662, which can be used as a model strain to study the ecological importance of effective alginate-degrading vibrios in the ocean. IMPORTANCE Alginate is an important carbon source in the marine environment, and vibrios are major alginate utilizers. Previous studies focused only on the characteristics of individual alginate lyases in vibrios, but few of them discussed the comprehensive alginate-degrading strategy. Here, we depicted the alginate utilization mechanism and its ecological implications of a highly efficient alginate-degrading Vibrio strain, WXL662, which contained 11 alginate lyases with distinct enzymatic characteristics. Importantly, unlike other vibrios with only one alginate utilization locus (AUL) or the previously reported "scattered" system, three AUL were identified in WXL662. Additionally, the involvement of outer membrane vesicles (OMVs) in the secretion of alginate lyases is proposed for the first time.

GSK-3ß inhibitor TDZD-8 prevents reduction of aquaporin-1 expression via activating autophagy under renal ischemia reperfusion injury.

Renal ischemia/reperfusion (I/R) injury is a main cause of acute kidney injury (AKI). Aquaporin (AQP)-1 water channel in the kidney is critical for the maintenance of water homeostasis and the urinary concentrating ability. Increasing evidence supports an important role of autophagy in the pathogenesis of AKI induced by renal I/R. The purpose of the present study is to investigate whether activation of autophagy prevents downregulation of AQP1 protein induced by renal I/R and potential molecular mechanisms. Renal I/R induced consistently reduced protein expression of AQP1, 2, and 3, as well as sodium cotransporters Na+ -K+ -2Cl- cotransporter and α-Na,K-ATPase, which was associated with increased urine output and decreased creatinine clearance in rats. Renal I/R also suppressed autophagy and increased inflammatory responses in the kidney. 4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), the glycogen synthase kinase-3ß inhibitor, ameliorated renal injury under I/R, activated autophagy and markedly increased expression of AQPs and sodium transporters in the kidney, which was associated with improved urine output and creatinine clearance in rats. Hypoxia/reoxygenation (H/R) induced suppression of autophagy and downregulation of AQP1 in murine inner medullary collecting duct 3 (IMCD3) cells, which was fully prevented by TDZD-8 treatment. Inhibition of autophagy by 3-methyladenine or Atg5 gene knockdown attenuated recovery of AQP1 protein expression induced by TDZD-8 in IMCD3 cells with H/R. Interleukin-1 beta (IL-1ß) decreased the abundance of AQP1 protein in IMCD3 cells. H/R induced increases in protein expression of nod-like receptor pyrin domain-containing 3 and IL-1ß, which was reversed by TDZD-8. In conclusion, TDZD-8 treatment prevented downregulation of AQP1 expression under renal I/R injury, likely via activating autophagy and decreasing IL-1ß production.

Biochemical Characterization of a Novel Endo-1,3-ß-Glucanase from the Scallop Chlamys farreri.

Endo-1,3-ß-glucanases derived from marine mollusks have attracted much attention in recent years because of their unique transglycosylation activity. In this study, a novel endo-1,3-ß-glucanase from the scallop Chlamys farreri, named Lcf, was biochemically characterized. Unlike in earlier studies on marine mollusk endo-1,3-ß-glucanases, Lcf was expressed in vitro first. Enzymatic analysis demonstrated that Lcf preferred to hydrolyze laminarihexaose than to hydrolyze laminarin. Furthermore, Lcf was capable of catalyzing transglycosylation reactions with different kinds of glycosyl acceptors. More interestingly, the transglycosylation specificity of Lcf was different from that of other marine mollusk endo-1,3-ß-glucanases, although they share a high sequence identity. This study enhanced our understanding of the diverse enzymatic specificities of marine mollusk endo-1,3-ß-glucanases, which facilitated development of a unique endo-1,3-ß-glucanase tool in the synthesis of novel glycosides.

Chitosan Oligosaccharide Ameliorates Nonalcoholic Fatty Liver Disease (NAFLD) in Diet-Induced Obese Mice.

Nonalcoholic fatty liver disease (NAFLD) is a global epidemic, and there is no standard and efficient therapy for it. Chitosan oligosaccharide (COS) is widely known to have various biological effects, and in this study we aimed to evaluate the liver-protective effect in diet-induced obese mice for an enzymatically digested product of COS called COS23 which is mainly composed of dimers and trimers. An integrated analysis of the lipidome and gut microbiome were performed to assess the effects of COS23 on lipids in plasma and the liver as well as on intestinal microbiota. Our results revealed that COS23 obviously attenuated hepatic steatosis and ameliorated liver injury in diet-induced obese mice. The hepatic toxic lipids-especially triglycerides (TGs) and free fatty acids (FFAs)-were decreased dramatically after COS23 treatment. COS23 regulated lipid-related pathways, especially inhibiting the expressions of FFA-synthesis-related genes and inflammation-related genes. Furthermore, COS23 could alter lipid profiles in plasma. More importantly, COS23 also decreased the abundance of Mucispirillum and increased the abundance of Coprococcus in gut microbiota and protected the intestinal barrier by up-regulating the expression of tight-junction-related genes. In conclusion, COS23, an enzymatically digested product of COS, might serve as a promising candidate in the clinical treatment of NAFLD.

Structural and biochemical characterization of a multidomain alginate lyase reveals a novel role of CBM32 in CAZymes.

Noncatalytic carbohydrate binding modules (CBMs) have been demonstrated to play various roles with cognate catalytic domains. However, for polysaccharide lyases (PLs), the roles of CBMs remain mostly unknown. AlyB is a multidomain alginate lyase that contains CBM32 and a PL7 catalytic domain. The AlyB structure determined herein reveals a noncanonical alpha helix linker between CBM32 and the catalytic domain. More interestingly, CBM32 and the linker does not significantly enhance the catalytic activity but rather specifies that trisaccharides are predominant in the degradation products. Detailed mutagenesis, biochemical and cocrystallization analyses show "weak but important" CBM32 interactions with alginate oligosaccharides. In combination with molecular modeling, we propose that the CBM32 domain serves as a "pivot point" during the trisaccharide release process. Collectively, this work demonstrates a novel role of CBMs in the activity of the appended PL domain and provides a new avenue for the well-defined generation of alginate oligosaccharides by taking advantage of associated CBMs.

Characterization of a Novel PolyM-Preferred Alginate Lyase from Marine Vibrio splendidus OU02.

Alginate lyases are enzymes that degrade alginate into oligosaccharides which possess a variety of biological activities. Discovering and characterizing novel alginate lyases has great significance for industrial and medical applications. In this study, we reported a novel alginate lyase, AlyA-OU02, derived from the marine Vibrio splendidus OU02. The BLASTP searches showed that AlyA-OU02 belonged to polysaccharide lyase family 7 (PL7) and contained two consecutive PL7 domains, which was rare among the alginate lyases in PL7 family. Both the two domains, AlyAa and AlyAb, had lyase activities, while AlyAa exhibited polyM preference, and AlyAb was polyG-preferred. In addition, the enzyme activity of AlyAa was much higher than AlyAb at 25 °C. The full-length enzyme of AlyA-OU02 showed polyM preference, which was the same as AlyAa. AlyAa degraded alginate into di-, tri-, and tetra-alginate oligosaccharides, while AlyAb degraded alginate into tri-, tetra-, and penta-alginate oligosaccharides. The degraded products of AlyA-OU02 were similar to AlyAa. Our work provided a potential candidate in the application of alginate oligosaccharide production and the characterization of the two domains might provide insights into the use of alginate of this organism.

Structural and biochemical insights into the degradation mechanism of chitosan by chitosanase OU01.

BACKGROUND: A detailed knowledge about the degradation mechanism of chitosanase hydrolysis is critical for the design of novel enzymes to produce well-defined chito-oligosaccharide products. METHODS: Through the combination of structural and biochemical analysis, we present new findings that provide novel insights into the degradation mechanism of chitosanase OU01. RESULTS: We have determined the crystal structure of Asp(43)/Ala mutant of OU01, and have trapped the hydrolyzed product of the reaction. This structure reveals the role of the general acid (Glu(25)) in catalysis. Two structural features about the mechanisms of the non-processive chitosanases are described for the first time. 1). Structural comparison reveals that the enzyme goes through an open-closed-open conformational transition upon substrate binding and product release; 2). polar residues constitute the substrate binding cleft. Additional site important for polymeric substrate recognition is identified and a three-step polymeric substrate recognition mechanism is proposed. CONCLUSIONS: Detailed substrate recognition mechanism is described for non-processive chitosanase for the first time. GENERAL SIGNIFICANCE: These findings provide new structural insights into the understanding of overall hydrolysis mechanism for non-processive chitosanase, and also will facilitate the design of new enzymes used for industrial purpose.

Structural insights into the substrate-binding mechanism for a novel chitosanase.

Chitosanase is able to specifically cleave ß-1,4-glycosidic bond linkages in chitosan to produce a chito-oligomer product, which has found a variety of applications in many areas, including functional food and cancer therapy. Although several structures for chitosanase have been determined, the substrate-binding mechanism for this enzyme has not been fully elucidated because of the lack of a high-resolution structure of the chitosanase-substrate complex. In the present study we show the crystal structure of a novel chitosanase OU01 from Microbacterium sp. in complex with its substrate hexa-glucosamine (GlcN)6, which belongs to the GH46 (glycoside hydrolyase 46) family in the Carbohydrate Active Enzymes database (http://www.cazy.org/). This structure allows precise determination of the substrate-binding mechanism for the first time. The chitosanase-(GlcN)6 complex structure demonstrates that, from the -2 to +1 position of the (GlcN)6 substrate, the pyranose rings form extensive interactions with the chitosanase-binding cleft. Several residues (Ser27, Tyr37, Arg45, Thr58, Asp60, His203 and Asp235) in the binding cleft are found to form important interactions required to bind the substrate. Site-directed mutagenesis of these residues showed that mutations of Y37F and H203A abolish catalytic activity. In contrast, the mutations T58A and D235A only lead to a moderate loss of catalytic activity, whereas the S27A mutation retains ~80% of the enzymatic activity. In combination with previous mutagenesis studies, these results suggest that the -2, -1 and +1 subsites play a dominant role in substrate binding and catalysis. DSF (differential scanning fluorimetry) assays confirmed that these mutations had no significant effect on protein stability. Taken together, we present the first mechanistic interpretation for the substrate (GlcN)6 binding to chitosanase, which is critical for the design of novel chitosanase used for biomass conversion.

Fasting-induced miR-7a-5p in AgRP neurons regulates food intake.

OBJECTIVE: The molecular control of feeding after fasting is essential for maintaining energy homeostasis, while overfeeding usually leads to obesity. Identifying non-coding microRNAs (miRNAs) that control food intake could reveal new oligonucleotide-based therapeutic targets for treating obesity and its associated diseases. This study aims to identify a miRNA modulating food intake and its mechanism in neuronal regulation of food intake and energy homeostasis. METHODS: A comprehensive genome-wide miRNA screening in the arcuate nucleus of the hypothalamus (ARC) of fasted mice and ad libitum mice was performed. Through stereotactic virus injections, intracerebroventricular injections, and miRNA sponge technology, miR-7a-5p was inhibited specifically in AgRP neurons and the central nervous system, and metabolic phenotypes were monitored. Quantitative real-time PCR, Western blotting, immunofluorescence, whole-cell patch-clamp recording, and luciferase reporter assay were used to investigate the mechanisms underlying miR-7a-5p's regulation of food intake. RESULTS: We found a significant increase in miR-7a-5p levels after fasting. miR-7a-5p was highly expressed in the ARC, and inhibition of miR-7a-5p specifically in AgRP neurons reduced food intake and body weight gain. miR-7a-5p inhibited S6K1 gene expression by binding to its 3'-UTR. Furthermore, the knockdown of ribosomal S6 kinase 1 (S6K1) in AgRP neurons can partially reverse the effects caused by miR-7a-5p inhibition. Importantly, intracerebroventricular administration of the miR-7a-5p inhibitor could also reduce food intake and body weight gain. CONCLUSION: Our findings suggest that miR-7a-5p responds to energy deficit and regulates food intake by fine-tuning mTOR1/S6K1 signaling in the AgRP neurons, which could be a promising oligonucleotide-based therapeutic target for treating obesity and its associated diseases.

Well-defined alginate oligosaccharides ameliorate joint pain and inflammation in a mouse model of gouty arthritis.

Background: Gouty arthritis causes severe pain and inflammation. Alginate oligosaccharides (AOSs) are natural products derived from alginate and have anti-inflammatory properties. We explored the potential effects of AOSs with different degrees of polymerization (Dp) on gouty arthritis and associated mechanisms. Methods: We established a mouse model of gouty arthritis by injecting monosodium urate (MSU) into ankle joint. Nocifensive behavior, gait and ankle swelling were used to study AOS's effects. Biochemical assays, in vivo imaging, live cell Ca2+ imaging, electrophysiology, RNA-sequencing, etc. were used for mechanism exploration. Results: AOS2 (Dp=2), AOS3 (Dp=3) and AOS4 (Dp=4) all inhibited ankle swelling, whereas AOS2&3 produced the most obvious analgesia on model mice. AOS3, which was picked for further evaluation, produced dose-dependent ameliorative effects on model mice. AOS3 reversed gait impairments but did not alter locomotor activity. AOS3 inhibited NLRP3 inflammasome activation and inflammatory cytokine up-regulation in ankle joint. AOS3 ameliorated MSU-induced oxidative stress and reactive oxygen species (ROS) production both in vivo and in vitro and reversed the impaired mitochondrial bioenergetics. AOS3 activated the Nrf2 pathway and promoted Nrf2 disassociation from Keap1-bound complex and Nrf2 nuclear translocation, thus facilitating antioxidant gene expression via Nrf2-dependent mechanism. Nrf2 gene deficiency abolished AOS3's ameliorative effects on pain, inflammation and oxidative stress in ankle joints of model mice. AOS3 reduced TRPV1 functional enhancement in DRG neurons and constrained neuroactive peptide release. Conclusions: AOS3 ameliorates gouty arthritis via activating Nrf2-dependent antioxidant signaling, resulting in suppression of ROS-mediated NLRP3 inflammasome activation and TRPV1 enhancement. AOS3 may be novel therapeutics for gouty arthritis.

Determination of oligosaccharide product distributions of PL7 alginate lyases by their structural elements.

Alginate lyases can be used to produce well-defined alginate oligosaccharides (AOSs) because of their specificities for AOS products. A large number of alginate lyases have been recorded in the CAZy database; however, the majority are annotated-only alginate lyases that include little information on their products, thus limiting their applications. Here, we establish a simple and experiment-saving approach to predict product distributions for PL7 alginate lyases through extensive structural biology, bioinformatics and biochemical studies. Structural study on several PL7 alginate lyases reveals that two loops around the substrate binding cleft determine product distribution. Furthermore, a database containing the loop information of all annotated-only single-domain PL7 alginate lyases is constructed, enabling systematic exploration of the association between loop and product distribution. Based on these results, a simplified loop/product distribution relationship is proposed, giving us information on product distribution directly from the amino acid sequence.

Sulfated alginate oligosaccharide exerts antitumor activity and autophagy induction by inactivating MEK1/ERK/mTOR signaling in a KSR1-dependent manner in osteosarcoma.

Alginate oligosaccharide (AOS) has the function to inhibit tumor progression and the sulfated modification can enhance the antitumor activity. To date, the function and mechanism of sulfated AOS (AOS-SO4) in tumors remain largely elusive. We prepared AOS by the enzymatic degradation of alginate, collected AOS-SO4 by sulfating following the canonical procedure. Using these materials, in vitro assays showed that both AOS and AOS-SO4 elicited antitumor effects in osteosarcoma cells. Sulfated modification significantly enhanced the antitumor activity. In addition, AOS-SO4 had obvious effects on cell cycle arrest, apoptosis, and autophagy induction in vitro and in vivo. Mechanistically, we observed that AOS-SO4 treatment triggered proapoptotic autophagy by inhibiting MEK1/ERK/mTOR signaling. The ERK activator reversed AOS-SO4-induced autophagy. More importantly, we found that KSR1 interacted with MEK1 and functioned as a positive regulator of MEK1 protein in osteosarcoma cells. High KSR1 expression was significantly associated with poor survival in osteosarcoma patients. Together, these results suggest that AOS-SO4 has a better antitumor effect in osteosarcoma by inhibiting MEK1/ERK/mTOR signaling, which is KSR1-dependent; thus, AOS-SO4 can be a new potential therapeutic candidate for the treatment of osteosarcoma.

Structural insights into the substrate-binding cleft of AlyF reveal the first long-chain alginate-binding mode.

The products of alginate degradation, alginate oligosaccharides (AOS), have potential applications in many areas, including functional foods and marine drugs. Enzyme-based approaches using alginate lyases have advantages in the preparation of well defined AOS and have attracted much attention in recent years. However, a lack of structural insight into the whole substrate-binding cleft for most known alginate lyases severely hampers their application in the industrial generation of well defined AOS. To solve this issue, AlyF was co-crystallized with the long alginate oligosaccharide G6 (L-hexaguluronic acid hexasodium salt), which is the longest bound substrate in all solved alginate lyase complex structures. AlyF formed interactions with G6 from subsites -3 to +3 without additional substrate-binding site interactions, suggesting that the substrate-binding cleft of AlyF was fully occupied by six sugars, which was further confirmed by isothermal titration calorimetry and differential scanning calorimetry analyses. More importantly, a combination of structural comparisons and mutagenetic analyses determined that three key loops (loop 1, Lys215-Glu236; loop 2, Gln402-Ile416; loop 3, Arg334-Gly348) mainly function in binding long substrates (degree of polymerization of >4). The potential flexibility of loop 1 and loop 2 might enable the substrate to continue to enter the cleft after binding to subsites +1 to +3; loop 3 stabilizes and orients the substrate at subsites -2 and -3. Taken together, these results provide the first possible alginate lyase-substrate binding profile for long-chain alginates, facilitating the rational design of new enzymes for industrial purposes.

Substrate-Binding Mode and Intermediate-Product Distribution Coguided Protein Design of Alginate Lyase AlyF for Altered End-Product Distribution.

Recently, we reported alginate lyase AlyF that predominantly produced trisaccharides (the trisaccharide content is 87.0%), and the determination of its substrate-binding mode facilitated its protein engineering for new product distribution. To clarify the relationship between the substrate-binding pocket and end-product distribution, the open binding pocket change was initially designed. The resulting F128T_W172R mutant of AlyF exhibited different intermediate-product distributions but still similar end-product distributions. However, these observations suggested that cleavage pattern changes for intermediate products might contribute to an altered end-product distribution. Structural analysis indicated that the sugar-binding affinity at subsite -2 should be redesigned to achieve this goal. Thus, residue Arg266, which is involved in sugar binding at subsite -2, was selected for site-saturation mutagenesis in the F128T_W172R mutant. The dominant end products of the F128T_W172R_R226H mutant were altered to disaccharides and trisaccharides (the disaccharide content increased to 40.5%).

Hyoid-complex elevation and stimulation technique restores swallowing function in patients with lateral medullary syndrome: Two case reports.

BACKGROUND: A swallowing disorder may occur following a brainstem stroke, especially one that occurs in the swallowing centers. Lateral medullary syndrome (referred to as LMS), a rare condition in which a vascular event occurs in the territory of the posterior inferior cerebellar artery or the vertebral artery, has been reported to lead to more severe and longer lasting dysphagia. CASE SUMMARY: We report two patients with dysphagia due to LMS and propose a novel technique named hyoid-complex elevation and stimulation technique (known as HEST). The two patients had no other functional incapacity back into life, but nasogastric feeding was the only possible way for nutrition because of severe aspirations. Swallowing function was evaluated by functional oral intake scale, modified water swallow test, surface electromyographic signal associated with video fluorography swallowing study to assess the situation of aspiration, pharyngeal residue, pharyngeal peristalsis, upper esophageal opening and the ability of deglutition. Both patients were treated with the HEST method for dysphagia and recovered quickly. CONCLUSION: HEST is effective for shortening the in-hospital time and improving the quality of life for patients with dysphagia who suffer from LMS and likely other strokes.

Brain hothubs and dark functional networks: correlation analysis between amplitude and connectivity for Broca's aphasia.

Source localization and functional brain network modeling are methods of identifying critical regions during cognitive tasks. The first activity estimates the relative differences of the signal amplitudes in regions of interest (ROI) and the second activity measures the statistical dependence among signal fluctuations. We hypothesized that the source amplitude-functional connectivity relationship decouples or reverses in persons having brain impairments. Five Broca's aphasics with five matched cognitively healthy controls underwent overt picture-naming magnetoencephalography scans. The gamma-band (30-45 Hz) phase-locking values were calculated as connections among the ROIs. We calculated the partial correlation coefficients between the amplitudes and network measures and detected four node types, including hothubs with high amplitude and high connectivity, coldhubs with high connectivity but lower amplitude, non-hub hotspots, and non-hub coldspots. The results indicate that the high-amplitude regions are not necessarily highly connected hubs. Furthermore, the Broca aphasics utilized different hothub sets for the naming task. Both groups had dark functional networks composed of coldhubs. Thus, source amplitude-functional connectivity relationships could help reveal functional reorganizations in patients. The amplitude-connectivity combination provides a new perspective for pathological studies of the brain's dark functional networks.

Structural insights into a novel Ca2+-independent PL-6 alginate lyase from Vibrio OU02 identify the possible subsites responsible for product distribution.

Alginate lyases have a wide range of industrial applications, such as oligosaccharide preparation, medical treatment, and bioconversion. Therefore, the discovery and characterization of novel alginate lyases are extremely important. PL-6 alginate lyases are classified into two groups: those with a single domain or two domains. However, only one structure of a two-domain alginate lyase has been determined to date. In this study, we characterized a novel single-domain PL-6 alginate lyase (named AlyF). According to the biochemical analysis, AlyF possesses unique features compared with other PL-6 enzymes, including (1) a Ca2+-independent catalytic mechanism and (2) a PolyG-specific cleavage specificity that predominantly produces trisaccharides. The structures of AlyF and its complexes described here reveal the structural basis for these unique features and substrate binding mechanisms, which were further confirmed using mutagenesis. More importantly, we determined the possible subsites specifying the predominantly trisaccharide products of AlyF, which may facilitate the rational design of AlyF for potential applications in preparing a single alginate oligomer.

The discovered chimeric protein plays the cohesive role to maintain scallop byssal root structural integrity.

Adhesion is essential for many marine sessile organisms. Unraveling the compositions and assembly of marine bioadheisves is the fundamental to understand their physiological roles. Despite the remarkable diversity of animal bioadhesion, our understanding of this biological process remains limited to only a few animal lineages, leaving the majority of lineages remain enigmatic. Our previous study demonstrated that scallop byssus had distinct protein composition and unusual assembly mechanism apart from mussels. Here a novel protein (Sbp9) was discovered from the key part of the byssus (byssal root), which contains two Calcium Binding Domain (CBD) and 49 tandem Epidermal Growth Factor-Like (EGFL) domain repeats. Modular architecture of Sbp9 represents a novel chimeric gene family resulting from a gene fusion event through the acquisition of CBD2 domain by tenascin like (TNL) gene from Na+/Ca2+ exchanger 1 (NCX1) gene. Finally, free thiols are present in Sbp9 and the results of a rescue assay indicated that Sbp9 likely plays the cohesive role for byssal root integrity. This study not only aids our understanding of byssus assembly but will also inspire biomimetic material design.

Characterization of an Atypical Metalloproteinase Inhibitors Like Protein (Sbp8-1) From Scallop Byssus.

Adhesion is a vital physiological process for many marine molluscs, including the mussel and scallop, and therefore it is important to characterize the proteins involved in these adhesives. Although several mussel byssal proteins were identified and characterized, the study for scallop byssal proteins remains scarce. Our previous study identified two foot-specific proteins (Sbp7, Sbp8-1), which were annotated as the tissue inhibitors of metalloproteinases (TIMPs). Evolutionary analysis suggests that the TIMP genes of Chlamys farreri had gone through multiple gene duplications during evolution, and their potential functional roles in foot may have an ancient evolutionary origin. Focusing on the Sbp8-1, the sequence alignment and biochemical analyses suggest that Sbp8-1 is an atypical TIMP. One significant feature is the presence of two extra free Cys residues at its C-terminus, which causes the Sbp8-1 polymerization. Considering the fact that the no inhibitory activity was observed and it is mainly distributed in byssal thread and plaque, we proposed that this atypical Sbp8-1 may play as the cross-linker in scallop byssus. This study facilitates not only the understanding of scallop byssus assembly, also provides the inspiration of water-resistant materials design.