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Spider silks with excellent mechanical properties attract more attention from scientists worldwide, and the dragline silk that serves as the framework of the spider's web is considered one of the strongest fibers. However, it is unfeasible for large-scale production of spider silk due to its highly territorial, cannibalistic, predatory, and solitary behavior. Herein, to alleviate some of these problems and explore aneasy way to produce spider fibers, we constructed recombinant baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) simultaneously expressing Trichonephila clavipes native ampullate spidroin 2 (MaSp-G) and spidroin 1 (MaSp-C) driven by the promoters of silkworm fibroin genes, to infect the nonpermissive Bombyx mori larvae at the fifth instar. MaSp-G and MaSp-C were co-expressed in the posterior silk glands (PSGs) of infected silkworms and successfully secreted into the lumen of the silk gland for fibroin globule assembly. The integration of MaSp-G and MaSp-C into silkworm silk fibers significantly improved the mechanical properties of these chimeric silk fibers, especially the strength and extensibility, which may be caused by the increment of β-sheet in the chimeric silkworm/spider silk fiber. These results demonstrated that silkworms could be developed as the nonpermissive heterologous host for the mass production of chimeric silkworm/spider silk fibers via the recombinant baculovirus AcMNPV., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

The stable energy barrier of mucin and soy hull polysaccharide (SHP) is established at the air/water interface in the intestinal fluid and is conducive to the absorption and transportation of nutrients. This study aimed to investigate the effect of different concentrations (0.5 % and 1.5 %) of Na + and K + on the energy barrier through the digestive system model in vitro. The interaction between ions and microwave-assisted ammonium oxalate-extracted SP (MASP)/mucus was characterized by particle size, zeta potential, interfacial tension, surface hydrophobicity, Fourier transform infrared spectroscopy, endogenous fluorescence spectroscopy, microstructure, and shear rheology. The results showed that the interactions between ions and MASP/mucus included electrostatic interaction, hydrophobic interaction, and hydrogen bond. The MASP/mucus miscible system was destabilized after 12 h, and the ions could improve the system stability to some extent. MASP aggregated continuously with the increase in the ion concentration, and large MASP aggregates were trapped above the mucus layer. Furthermore, the adsorption of MASP/mucus at the interface increased and then decreased. These findings provided a theoretical basis for an in-depth understanding of the mechanism of action of MASP in the intestine., Competing Interests: Declaration of competing interest None., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Apart from autopsy, tissue correlates of coronavirus disease 2019 (COVID-19) clinical stage are lacking. In the current study, cutaneous punch biopsy specimens of 15 individuals with severe/critical COVID-19 and six with mild/moderate COVID-19 were examined. Evidence for arterial and venous microthrombi, deposition of C5b-9 and MASP2 (representative of alternative and lectin complement pathways, respectively), and differential expression of interferon type I-driven antiviral protein MxA (myxovirus resistance A) versus SIN3A, a promoter of interferon type I-based proinflammatory signaling, were assessed. Control subjects included nine patients with sepsis-related acute respiratory distress syndrome (ARDS) and/or acute kidney injury (AKI) pre-COVID-19. Microthrombi were detected in 13 (87%) of 15 patients with severe/critical COVID-19 versus zero of six patients with mild/moderate COVID-19 (P < 0.001) and none of the nine patients with pre-COVID-19 ARDS/AKI (P < 0.001). Cells lining the microvasculature staining for spike protein of severe acute respiratory syndrome coronavirus 2, the etiologic agent of COVID-19, also expressed tissue factor. C5b-9 deposition occurred in 13 (87%) of 15 patients with severe/critical COVID-19 versus zero of six patients with mild/moderate COVID-19 (P < 0.001) and none of the nine patients with pre-COVID-19 ARDS/AKI (P < 0.001). MASP2 deposition was also restricted to severe/critical COVID-19 cases. MxA expression occurred in all six mild/moderate versus two (15%) of 13 severe/critical cases (P < 0.001) of COVID-19. In contrast, SIN3A was restricted to severe/critical COVID-19 cases co-localizing with severe acute respiratory syndrome coronavirus 2 spike protein. SIN3A was also elevated in plasma of patients with severe/critical COVID-19 versus control subjects (P ≤ 0.02). In conclusion, the study identified premortem tissue correlates of COVID-19 clinical stage using skin. If validated in a longitudinal cohort, this approach could identify individuals at risk for disease progression and enable targeted interventions., (Copyright © 2022 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Unlabelled: ESSENTIALS: The lectin pathway's MASP-1/2 activates coagulation factors but the trigger of the activation is unknown. MASP-1/2 activation was assessed by quantifying complexes between MASPs and antithrombin/C1-inhibitor. Activated platelets and fibrin were demonstrated to activate MASP-1 and MASP-2 both in vitro and in vivo. These findings may represent a crossroad between the complement and the coagulation systems., Background: The activated forms of the complement lectin pathway (LP) proteases MASP-1 and MASP-2 are able to cleave the coagulation factors prothrombin, fibrinogen, factor XIII and thrombin-activatable fibrinolysis inhibitor in vitro. In vivo studies also show that MASP-1 is involved in thrombogenesis., Objectives: To clarify the not yet identified mechanisms involved in triggering activation of the LP during thrombotic reactions., Methods: Novel sandwich-ELISAs for detection of complexes between MASP-1 or MASP-2 and the serpins C1 inhibitor (C1-INH) or antithrombin (AT), were used to specifically detect and quantify the activated forms of MASP-1 and MASP-2., Results: Activated platelets were shown by flow cytometry to bind Ficolin-1, -2 and -3 but not MBL, which was associated with activation of MASP-1 and MASP-2. We also demonstrated that fibrin and the plasmin-generated fibrin fragment DD in plasma, bind and activate MASP-1 and MASP-2. As demonstrated by the ELISA and SDS-PAGE/Western blotting, the fibrin-associated activation was reflected in a specific inactivation by AT during clotting without the assistance of heparin. In all other cases the MASPs were, as previously reported, inactivated by C1-INH. In systemic lupus erythematosus patients with thrombotic disease and in polytrauma patients, the levels of activated MASP-1 and MASP-2 in complex with both AT and C1-INH were associated with markers of thrombotic disease and contact/coagulation system activation., Conclusions: MASP-1 and MASP-2 are activated during blood clotting. This activation is triggered by activated platelets and by the generation of fibrin during thrombotic reactions in vitro and in vivo, and may represent a novel activation/amplification mechanism in thromboinflammation., (© 2015 International Society on Thrombosis and Haemostasis.)

A family of serine proteases mediates the proteolytic cascades of several defense mechanisms in vertebrates, such as the complement system, blood coagulation and fibrinolysis. These proteases usually form large complexes with other glycoproteins. Their common features are their modular structures and restricted substrate specificities. The lectin pathway of complement, where mannose-binding lectin (MBL) recognizes the carbohydrate structures on pathogens, is activated by mannose-binding lectin-associated serine protease-2 (MASP-2). We present the 2.25A resolution structure of the catalytic fragment of MASP-2 encompassing the second complement control protein module (CCP2) and the serine protease (SP) domain. The CCP2 module stabilizes the structure of the SP domain as demonstrated by differential scanning calorimetry measurements. The asymmetric unit contains two molecules with different CCP-SP domain orientations, reflecting increased modular flexibility at the CCP2/SP joint. This flexibility may partly explain the ability of the MASP-2 dimer to perform all of its functions alone, whereas the same functions are mediated by the much larger C1r2-C1s2 tetramer in the C1 complex of the classical pathway. The main scaffold of the MASP-2 SP domain is chymotrypsin-like. Eight surface loops determine the S1 and other subsite specificities. Surprisingly, some surface loops of MASP-2, e.g. loop 1 and loop 2, which form the S1 pocket are similar to those of trypsin, and show significant differences if compared with those of C1s, indicating that the nearly identical substrate specificities of C1s and MASP-2 are realized through different sets of enzyme-substrate interactions.

The lectin pathway is part of the innate immune system providing a first line of defence against infections. Mannan-binding lectin (MBL) and ficolins, in complex with MBL-associated serine proteases (MASPs), are capable of activating the complement system, thus mediating the destruction of infectious agents. MASP-2 cleaves C4 and C2 and is thus crucial for the activation of downstream complement components. We present an assay for quantifying total MASP-2 in plasma and serum samples. The assay is a sandwich type assay using a combination of two monoclonal anti-MASP-2 antibodies, one directed against the N-terminal part of MASP-2 and the other against its C-terminal part. Based on a population of Danish blood donors, the average MASP-2 concentration was estimated at 534 (S.D.+/-213) ng per ml of plasma. Characterization of the MASP-2 protein in serum showed high stability at 4 degrees C and at ambient temperature but a rapid decline at 37 degrees C. Gel permeation chromatography (GPC) indicated that all MASP-2 in serum is present in complexes with MBL and ficolins.

Background: Hakata antigen (Hakata) is a novel serum glycoprotein that consists of collagen- and fibrinogen-like domains, similar to ficolin/p35. Our research suggested that serum Hakata may be a target of a polysaccharide (PSA) produced by Aerococcus viridans., Methods: A. viridans was incubated with human plasma and Hakata-depleted plasma to examine Hakata binding and growth inhibition of A. viridans through binding with PSA., Results: When A. viridans was mixed with human acid citrate dextrose-one (ACD-A) plasma, it pulled down Hakata complexed with mannose-binding lectin (MBL)-associated serine proteases 1 and 2 (MASP-1 and MASP-2). This complex had the potential for C4 deposition. Serum Hakata circulates as Hakata-MASPs complex in the blood and is proteolytically active. By mixing A. viridans with human plasma, we prepared a Hakata-depleted plasma, deficient in Hakata-MASPs complex. This plasma failed to inhibit A. viridans growth plasma, but does not inhibit Staphylococcus aureus, Yersinia enterocolitica and Escherichia coli. However, a decrease of growth inhibition of A. viridans in Hakata-depleted plasma could be restored by adding a Hakata-MASPs complex preparation in a dose-dependent manner. On the other hand, the Hakata-MASPs complex exhibited strong binding to A. viridans, but not to S. aureus, Y. enterocolitica and E. coli., Conclusions: The serum concentration of Hakata is linked with growth inhibition of A. viridans upon binding of Hakata via PSA., (Copyright 2002 Elsevier Science B.V.)

Intravenous injection of a lipopolysaccharide (LPS) into mice induces a rapid accumulation of platelets in the lung and liver. When degradation of the accumulated platelets occurs, anaphylactoid shock follows rapidly, the severity of the shock paralleling the quantity of platelets accumulated in the lung. Here we examined the contributions made by LPS structure and the complement system to the platelet response to LPS. BALB/c mice were injected with an LPS from Escherichia coli O8, O9, O111, or K-12, or from recombinant mutants of K-12. The O-regions of the O8 and O9 LPSs consist of a mannose homopolysaccharide (MHP), while that of O111 consists of a heteropolysaccharide (not including mannose), and K-12 LPS lacks an O-region. O111 LPS was devoid of the ability to induce the platelet response or shock, while the ability of K-12 LPS was weak. The 2 recombinant LPSs-each having an O-region (from O8 or O9) linked to K-12 LPS-exhibited activities similar to or stronger than those of their original LPSs. Mannose-binding lectin (MBL) complexed with MBL-associated serine proteases (MASPs) bound strongly to LPSs containing MHP and caused C4 activation. Moreover, the abilities of these LPSs to activate the complement system corresponded well with their abilities to induce the platelet response and rapid shock. These results suggest that the structure of the O-antigen region is important for the platelet response to LPS, and that activation of the lectin pathway of the complement system is involved in this response.

A simple assay was developed to estimate functional mannose-binding lectin (MBL) levels in serum based on the principle of yeast-induced bystander lysis of chicken erythrocytes (ChE). The assay is sensitive to inhibition by ethylene glycol bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) (which allows alternative pathway activation), ethylene diamine tetraacetic acid (EDTA), mannose, N-acetylglucosamine and C1 esterase inhibitor (C1-INH), whereas it was not inhibited by galactose. A high-titer human anti-mannan antibody-containing serum with 0.06 microg MBL/ml gave a functional signal corresponding to 0.12 microg equivalents MBL/ml, indicating that anti-mannan antibodies are poorly hemolytic in the assay. The assay is well suited for the large-scale testing of patient samples for a functional MBL pathway of complement activation.

Ficolins are a group of proteins containing both a collagen-like domain and a fibrinogen-like domain and are found in varying tissues. Ficolins present in sera have a lectin activity toward N-acetylglucosamine through their fibrinogen-like domains. The domain organizations between ficolins and mannose-binding lectin (MBL) are very similar in that both consist of a collagen-like domain and a carbohydrate-binding domain, although their carbohydrate-binding moieties are different. MBL acts as an opsonin and activates complement in association with MBL-associated serine proteases (MASPs) and sMAP, truncated protein of MASP-2, via the lectin pathway. Like MBL, two types of human serum ficolins, L-ficolin/P35 and H-ficolin, are associated with MASPs and sMAP, and activate the lectin pathway. In addition, L-ficolin/P35 acts as an opsonin. These findings indicate that serum ficolins play an important a role in innate immunity in a similar manner to MBL.

Serum mannose-binding protein (MBP) is the first component of the lectin pathway of the complement cascade. It binds to sugars on the surface of pathogenic microorganisms and triggers complement fixation by activating an associated serine protease, designated MBP-associated serine protease-2 (MASP-2). Recent studies have provided insight into the interactions between MBP and MASP-2 that trigger complement activation. MBP/MASP complexes share many features with the C1 complex of the classical pathway. The relatively simple MBP/MASP complexes serve as useful models for understanding activation of the classical pathway of the complement cascade.

Mannan-binding lectin (MBL) activates complement independently of the adaptive, clonal immune system and thus presents an innate anti-microbial defence mechanism. Events in the MBL pathway of complement activation involve the binding of MBL to patterns of carbohydrate structures presented by the surface of micro-organisms. For the activation of complement to occur MBL must be associated with serine proteases (MBL-associated serine proteases, MASPs) in an MBL/MASP complex. When bound to micro-organisms, the MBL complex mediates the activation of C4 and C2, generating the C3 convertase, C4bC2b. The C4/C2 cleaving activity of the MBL complex is shared with the C1 complex of the classical pathway of complement activation. Different assays that allow for determination of the activity of the MBL complex in serum samples have been developed and are discussed in this report. We present data from one such assay (MBL/MASP activity test), which we have found useful for the routine evaluation of clinical samples. In this assay any influence of the classical pathway has been eliminated by using a hypertonic buffer, which inhibits the binding of C1q to immuncomplexes and disrupt the C1 complex, while leaving the function of the MBL complex intact. In parallel we determine the MBL concentration in the sample. As predicted a very high correlation is observed between the results of the two assays.

Mannan-binding lectin (MBL) and ficolins (L-ficolin and H-ficolin) initiate the lectin pathway of complement activation upon binding to microbial carbohydrates. The activation is mediated by associated serine proteases, termed MASPs, since they were discovered as MBL-associated serine proteases. The MASP family comprises three serine proteases, MASP-1, MASP-2 and MASP-3 and a non-enzymatic protein, MAp19. The MASPs show identical domain structure, shared also with C1r and C1s. MASP-1 and MASP-3 are alternative splice products of a single gene, MASP1/3, and have identical A chains, whereas they have individual B chains, encompassing the serine protease domain. MASP2 and MAp19 are alternative splice products of the MASP-2 gene, with MAp19 consisting of the first two domains of MASP-2 plus additional four amino acid residues. MASP-2 is the protease responsible for activating C4 and C2 to generate the C3 convertase, C4bC2b. The biological function of the remaining three proteins has not yet been resolved.

The Mannose-binding lectin-associated serine proteases (MASPs) have been the subject of intensive research particularly over the past 10 years. First one, then two, and currently 3 MASPs have been characterized. Initially it was thought likely that the MBL + MASPs system would resemble very closely the C1 complex of the complement classical pathway, and that MASP1 and MASP2 would have similar activities to their classical pathway homologues C1r and C1s. MASP2 does certainly have similar activities to C1s, but MASP1 does not have the activities of either C1r or C1s. MASP1 has been thought to act on the complement system by cleaving C3 directly, but work with recombinant and purified native MASP1 shows that direct C3 cleavage by this protease is very slow, and may not be biologically significant. MASP1 and MASP2 appear not to have such a narrow specificity as C1r and C1s, and may have significant substrates other than complement proteins. As an example, MASP1 does cleave fibrinogen, releasing fibrinopeptide B (a chemotactic factor) and also cleaves and activates plasma transglutaminase (Factor XIII). These reactions are also relevant to defence against microorganisms, and may represent a biologically significant action of MASP1.

Mannan-binding lectin (MBL) activates the complement system through cleavage of C4 and C2. Until recently it was thought that only one serine protease in complex with MBL (MBL-associated serine protease, MASP) mediates complement activation, but with the finding of a second MBL-associated serine protease, MASP-2, the activation process appears more elaborate, possibly resembling that of the C1 complex. The two MASPs share the domain organisation of C1r and C1s and it may be speculated that interaction between the two MASPs is required for complement activation in the same manner as with the C1 proteases. We have demonstrated that MASP-2 is a C4 cleaving component of the MBL/MASP complex. By analogy, one may thus speculate that, upon binding of MBL to carbohydrate, MASP-1 autoactivates and then activates MASP-2, but there is as yet no evidence for this. The components of C1 are present in serum in approximately equimolar amounts, whereas MASP-1 is in large excess over MBL. Pairwise comparison of the four proteases shows the primary structures to be approximately 40% identical. Phylogenetic analysis indicates that MASP-2 is closer to C1r and C1s than is MASP-1, but no particular association between MASP-2 and the C4 cleaving enzyme, C1s, can be deduced from sequence comparison.

Serum mannose-binding lectin binds to pathogens in association with a serine protease termed MASP, and in this form, plays a crucial role in innate immunity by activating complement in a manner similar to activation via the classical pathway. MASP, C1r and C1s belong to the same family of serine proteases. In addition to its presence in advanced species, MASP also exists in primitive life forms such as tunicates and may be an evolutionary prototype of this family.

We have previously shown that a novel C1s-like serine protease termed MBP-associated serine protease (MASP) is responsible for activation of the complement cascade initiated by mannose-binding protein (MBP). In this communication, we report that MASP is unique in having the proteolytic capacity to cleave C3 with subsequent activation of the alternative pathway, a capacity which C1s lacks.

The Ra-reactive factor (RaRF) found in vertebrate sera activates the C4 and C2 components of complement. The C4/C2-activating component of mouse RaRF has been found to contain a 100-kDa serine protease called P100. In the present study, we cloned a cDNA with cDNA of mouse RaRF P100 as a probe from a human liver cDNA library. An open reading frame of 2097 nucleotides encoding a protein of 699 residues was found in the cloned cDNA of 4489 nucleotides. This protein exhibits 87.4% amino acid homology with mouse P100, and 36.4% and 37.1% homologies with that of the C1r and C1s subcomponents of human complement, respectively. The characteristic nodules and domain of C1r and C1s were highly conserved in this protein. This indicates that the P100, together with the C1r and C1s, forms a unique protein family having the same module/domain constitution.