A novel ATG4B antagonist inhibits autophagy and has a negative impact on osteosarcoma tumors

Autophagy has been implicated in the progression and chemoresistance of various cancers. In this study, we have shown that osteosarcoma Saos-2 cells lacking ATG4B, a cysteine proteinase that activates LC3B, are defective in autophagy and fail to form tumors in mouse models. By combining in silico docking with in vitro and cell-based assays, we identified small compounds that suppressed starvation-induced protein degradation, LC3B lipidation, and formation of autophagic vacuoles. NSC185058 effectively inhibited ATG4B activity in vitro and in cells while having no effect on MTOR and PtdIns3K activities. In addition, this ATG4B antagonist had a negative impact on the development of Saos-2 osteosarcoma tumors in vivo. We concluded that tumor suppression was due to a reduction in ATG4B activity, since we found autophagy suppressed within treated tumors and the compound had no effects on oncogenic protein kinases. Our findings demonstrate that ATG4B is a suitable anti-autophagy target and a promising therapeutic target to treat osteosarcoma.     

Comparative analyses of ubiquitin-like ATG8 and cysteine protease ATG4 autophagy genes in the plant lineage and cross-kingdom processing of ATG8 by ATG4

Autophagy is important for degradation and recycling of intracellular components. In a diversity of genera and species, orthologs and paralogs of the yeast Atg4 and Atg8 proteins are crucial in the biogenesis of double-membrane autophagosomes that carry the cellular cargoes to vacuoles and lysosomes. Although many plant genome sequences are available, the ATG4 and ATG8 sequence analysis is limited to some model plants. We identified 28 ATG4 and 116 ATG8 genes from the available 18 different plant genome sequences. Gene structures and protein domain sequences of ATG4 and ATG8 are conserved in plant lineages. Phylogenetic analyses classified ATG8s into 3 subgroups suggesting divergence from the common ancestor. The ATG8 expansion in plants might be attributed to whole genome duplication, segmental and dispersed duplication, and purifying selection. Our results revealed that the yeast Atg4 processes Arabidopsis ATG8 but not human LC3A (HsLC3A). In contrast, HsATG4B can process yeast and plant ATG8s in vitro but yeast and plant ATG4s cannot process HsLC3A. Interestingly, in Nicotiana benthamiana plants the yeast Atg8 is processed compared to HsLC3A. However, HsLC3A is processed when coexpressed with HsATG4B in plants. Molecular modeling indicates that lack of processing of HsLC3A by plant and yeast ATG4 is not due to lack of interaction with HsLC3A. Our in-depth analyses of ATG4 and ATG8 in the plant lineage combined with results of cross-kingdom ATG8 processing by ATG4 further support the evolutionarily conserved maturation of ATG8. Broad ATG8 processing by HsATG4B and lack of processing of HsLC3A by yeast and plant ATG4s suggest that the cross-kingdom ATG8 processing is determined by ATG8 sequence rather than ATG4.     

AU4S: A novel synthetic peptide to measure the activity of ATG4 in living cells

ATG4 plays a key role in autophagy induction, but the methods for monitoring ATG4 activity in living cells are limited. Here we designed a novel fluorescent peptide named AU4S for noninvasive detection of ATG4 activity in living cells, which consists of the cell-penetrating peptide (CPP), ATG4-recognized sequence “GTFG,” and the fluorophore FITC. Additionally, an ATG4-resistant peptide AG4R was used as a control. CPP can help AU4S or AG4R to penetrate cell membrane efficiently. AU4S but not AG4R can be recognized and cleaved by ATG4, leading to the change of fluorescence intensity. Therefore, the difference between AU4S- and AG4R-measured fluorescence values in the same sample, defined as “F-D value,” can reflect ATG4 activity. By detecting the F-D values, we found that ATG4 activity paralleled LC3B-II levels in rapamycin-treated cells, but neither paralleled LC3B-II levels in starved cells nor presented a correlation with LC3B-II accumulation in WBCs from healthy donors or leukemia patients. However, when DTT was added to the system, ATG4 activity not only paralleled LC3B-II levels in starved cells in the presence or absence of autophagy inhibitors, but also presented a positive correlation with LC3B-II accumulation in WBCs from leukemia patients (R² = 0.5288). In conclusion, this study provides a convenient, rapid, and quantitative method to monitor ATG4 activity in living cells, which may be beneficial to basic and clinical research on autophagy.     

三白草中ATG4B抑制剂的发现及活性测定

验证从三白草中提取的两个化合物XGN56和XGN59对自噬关键蛋白ATG4B酶活性的影响及对自噬的调节作用。分子对接的方法验证化合物与游离ATG4B及ATG4B-LC3复合体的氢键结合作用;SDS-PAGE法及荧光共振能量转移法(FRET)测定化合物(10μmol/L)抑制ATG4B的IC50值;LC3融合GFP荧光标签检测化合物(10μmol/L)对LC3荧光聚集的影响,并设置正常组、给药组和药物联用Baf(0.5μmol/L)组;过表达GFP-LC3的WT-MEF及ATG5-/--MEF细胞检测化合物诱导LC3荧光点的情况。结果显示,XGN56和XGN59能分别与游离ATG4B和ATG4B-LC3复合体形成氢键作用,且两者均能剂量依赖地抑制ATG4B的酶切活性,体外IC50分别为7.74μmol/L和8.00μmol/L,同时能够ATG5依赖地促进GFP标记的自噬体的生成(P<0.001)。结果表明,两个化合物可能是通过一定程度地抑制ATG4B的酶活性从而促进细胞自噬水平。     

Essential role for the ATG4B protease and autophagy in bleomycin-induced pulmonary fibrosis

Autophagy is a critical cellular homeostatic process that controls the turnover of damaged organelles and proteins. Impaired autophagic activity is involved in a number of diseases, including idiopathic pulmonary fibrosis suggesting that altered autophagy may contribute to fibrogenesis. However, the specific role of autophagy in lung fibrosis is still undefined. In this study, we show for the first time, how autophagy disruption contributes to bleomycin-induced lung fibrosis in vivo using an Atg4b-deficient mouse as a model. Atg4b-deficient mice displayed a significantly higher inflammatory response at 7 d after bleomycin treatment associated with increased neutrophilic infiltration and significant alterations in proinflammatory cytokines. Likewise, we found that Atg4b disruption resulted in augmented apoptosis affecting predominantly alveolar and bronchiolar epithelial cells. At 28 d post-bleomycin instillation Atg4b-deficient mice exhibited more extensive and severe fibrosis with increased collagen accumulation and deregulated extracellular matrix-related gene expression. Together, our findings indicate that the ATG4B protease and autophagy play a crucial role protecting epithelial cells against bleomycin-induced stress and apoptosis, and in the regulation of the inflammatory and fibrotic responses.     

ATG4B contains a C-terminal LIR motif important for binding and efficient cleavage of mammalian orthologs of yeast Atg8

The cysteine protease ATG4B cleaves off one or more C-terminal residues of the inactive proform of proteins of the ortholog and paralog LC3 and GABARAP subfamilies of yeast Atg8 to expose a C-terminal glycine that is conjugated to phosphatidylethanolamine during autophagosome formation. We show that ATG4B contains a C-terminal LC3-interacting region (LIR) motif important for efficient binding to and cleavage of LC3 and GABARAP proteins. We solved the crystal structures of the GABARAPL1-ATG4B C-terminal LIR complex. Analyses of the structures and in vitro binding assays, using specific point mutants, clearly showed that the ATG4B LIR binds via electrostatic-, aromatic HP1 and hydrophobic HP2 pocket interactions. Both these interactions and the catalytic site-substrate interaction contribute to binding between LC3s or GABARAPs and ATG4B. We also reveal an unexpected role for ATG4B in stabilizing the unlipidated forms of GABARAP and GABARAPL1. In mouse embryonic fibroblast (MEF) atg4b knockout cells, GABARAP and GABARAPL1 were unstable and degraded by the proteasome. Strikingly, the LIR motif of ATG4B was required for stabilization of the unlipidated forms of GABARAP and GABARAPL1 in cells.     

TRAPPC11 functions in autophagy by recruiting ATG2B‐WIPI4/WDR45 to preautophagosomal membranes

TRAPPC11 has been implicated in membrane traffic and lipid‐linked oligosaccharide synthesis, and mutations in TRAPPC11 result in neuromuscular and developmental phenotypes. Here, we show that TRAPPC11 has a role upstream of autophagosome formation during macroautophagy. Upon TRAPPC11 depletion, LC3‐positive membranes accumulate prior to, and fail to be cleared during, starvation. A proximity biotinylation assay identified ATG2B and its binding partner WIPI4/WDR45 as TRAPPC11 interactors. TRAPPC11 depletion phenocopies that of ATG2 and WIPI4 and recruitment of both proteins to membranes is defective upon reduction of TRAPPC11. We find that a portion of TRAPPC11 and other TRAPP III proteins localize to isolation membranes. Fibroblasts from a patient with TRAPPC11 mutations failed to recruit ATG2B‐WIPI4, suggesting that this interaction is physiologically relevant. Since ATG2B‐WIPI4 is required for isolation membrane expansion, our study suggests that TRAPPC11 plays a role in this process. We propose a model whereby the TRAPP III complex participates in the formation and expansion of the isolation membrane at several steps.      

ATG4B/autophagin-1 regulates intestinal homeostasis and protects mice from experimental colitis

The identification of inflammatory bowel disease (IBD) susceptibility genes by genome-wide association has linked this pathology to autophagy, a lysosomal degradation pathway that is crucial for cell and tissue homeostasis. Here, we describe autophagy-related 4B, cysteine peptidase/autophagin-1 (ATG4B) as an essential protein in the control of inflammatory response during experimental colitis. In this pathological condition, ATG4B protein levels increase in parallel with the induction of autophagy. Moreover, ATG4B expression is significantly reduced in affected areas of the colon from IBD patients. Consistently, atg4b^−/− mice present Paneth cell abnormalities, as well as an increased susceptibility to DSS-induced colitis. atg4b-deficient mice exhibit significant alterations in proinflammatory cytokines and mediators of the immune response to bacterial infections, which are reminiscent of those found in patients with Crohn disease or ulcerative colitis. Additionally, antibiotic treatments and bone marrow transplantation from wild-type mice reduced colitis in atg4b^−/− mice. Taken together, these results provided additional evidence for the importance of autophagy in intestinal pathologies and describe ATG4B as a novel protective protein in inflammatory colitis. Finally, we propose that atg4b-null mice are a suitable model for in vivo studies aimed at testing new therapeutic strategies for intestinal diseases associated with autophagy deficiency.     

Insights into links between autophagy and the ubiquitin system from the structure of LC3B bound to the LIR motif from the E3 ligase NEDD4

Members of the LC3/GABARAP family of ubiquitin‐like proteins function during autophagy by serving as membrane linked protein‐binding platforms. Their C‐termini are physically attached to membranes through covalent linkage to primary amines on lipids such as phosphatidylethanolamine, while their ubiquitin‐like fold domains bind “LIR” (LC3‐Interacting Region) sequences found within an extraordinarily diverse array of proteins including regulators of autophagy, adaptors that recruit ubiquitinated cargoes to be degraded, and even proteins controlling processes at membranes that are not associated with autophagy. Recently, LC3/GABARAP proteins were found to bind the ubiquitin E3 ligase NEDD4 to influence ubiquitination associated with autophagy in human cell lines. Here, we use purified recombinant proteins to define LC3B interactions with a specific LIR sequence from NEDD4, present a crystal structure showing atomic details of the interaction, and show that LC3B‐binding can steer intrinsic NEDD4 E3 ligase activity. The data provide detailed molecular insights underlying recruitment of an E3 ubiquitin ligase to phagophores during autophagy.     

Synthesis and evaluation of novel benzotropolones as Atg4B inhibiting autophagy blockers

Autophagy is an intracellular degradation/recycling pathway that provides nutrients and building blocks to cellular metabolism and keeps the cytoplasm clear of obsolete proteins and organelles. During recent years, dysregulated autophagy activity has been reported to be a characteristic of many different disease types, including cancer and neurodegenerative disorders. This has created a strong case for development of autophagy modulating compounds as potential treatments for these diseases. Inhibitors of autophagy have been proposed as a therapeutic intervention in, e.g., advanced cancer, and inhibiting the cysteine protease Atg4B has been put forward as a main strategy to block autophagy. We recently identified and demonstrated -both in vitro and in vivo - that compounds with a benzotropolone basic structure targeting Atg4B, can significantly slow down tumor growth and potentiate the effect of classical chemotherapy. In this study we report the synthesis and inhibition profile of new benzotropolone derivatives with additional structural modifications at 6 different positions. To obtain a solid inhibition profile, all compounds were evaluated on three levels, including two cell-based assays to confirm autophagy and intracellular Atg4B inhibition and an SDS-PAGE-based experiment to assess in vitro Atg4B affinity. Several molecules with a promising profile were identified.     

Silibinin,a novel chemokine receptor type 4 antagonist,inhibits chemokine ligand 12-induced migration in breast cancer cells

PurposeC-X-C chemokine receptor type 4 (CXCR4) signaling has been demonstrated to be involved in cancer invasion and migration; therefore, CXCR4 antagonist can serve as an anti-cancer drug by preventing tumor metastasis. This study aimed to identify the CXCR4 antagonists that could reduce and/or inhibit tumor metastasis from natural products.Methods and resultsAccording to the molecular docking screening, we reported here silibinin as a novel CXCR4 antagonist. Biochemical characterization showed that silibinin blocked chemokine ligand 12 (CXCL12)-induced CXCR4 internalization by competitive binding to CXCR4, therefore inhibiting downstream intracellular signaling. In human breast cancer cells MDA-MB-231, which expresses high levels of CXCR4, inhibition of CXCL12-induced chemomigration can be found under silibinin treatment. Overexpression of CXCL12 sensitized MDA-MB-231 cells to the inhibition of silibinin, which was abolished by CXCR4 knockdown. The inhibition of silibinin was also observed in MCF-7/CXCR4 cells rather than MCF-7 cells that express low level of CXCR4.ConclusionsOur work demonstrated that silibinin is a novel CXCR4 antagonist that may have potential therapeutic use for prevention of tumor metastasis.     

AKT-mediated phosphorylation of ATG4B impairs mitochondrial activity and enhances the Warburg effect in hepatocellular carcinoma cells

Phosphorylation is a major type of post-translational modification, which can influence the cellular physiological function. ATG4B, a key macroautophagy/autophagy-related protein, has a potential effect on the survival of tumor cells. However, the role of ATG4B phosphorylation in cancers is still unknown. In this study, we identified a novel phosphorylation site at Ser34 of ATG4B induced by AKT in HCC cells. The phosphorylation of ATG4B at Ser34 had little effect on autophagic flux, but promoted the Warburg effect including the increase of L-lactate production and glucose consumption, and the decrease of oxygen consumption in HCC cells. The Ser34 phosphorylation of ATG4B also contributed to the impairment of mitochondrial activity including the inhibition of F₁Fo-ATP synthase activity and the elevation of mitochondrial ROS in HCC cells. Moreover, the phosphorylation of ATG4B at Ser34 enhanced its mitochondrial location and the subsequent colocalization with F₁Fo-ATP synthase in HCC cells. Furthermore, recombinant human ATG4B protein suppressed the activity of F₁Fo-ATP synthase in MgATP submitochondrial particles from patient-derived HCC tissues in vitro. In brief, our results demonstrate for the first time that the phosphorylation of ATG4B at Ser34 participates in the metabolic reprogramming of HCC cells via repressing mitochondrial function, which possibly results from the Ser34 phosphorylation-induced mitochondrial enrichment of ATG4B and the subsequent inhibition of F₁Fo-ATP synthase activity. Our findings reveal a noncanonical working pattern of ATG4B under pathological conditions, which may provide a scientific basis for developing novel strategies for HCC treatment by targeting ATG4B and its Ser34 phosphorylation.     

The 1:2 complex between RavZ and LC3 reveals a mechanism for deconjugation of LC3 on the phagophore membrane

Hosts utilize macroautophagy/autophagy to clear invading bacteria; however, bacteria have also developed a specific mechanism to survive by manipulating the host cell autophagy mechanism. One pathogen, Legionella pneumophila, can hinder host cell autophagy by using the specific effector protein RavZ that cleaves phosphatidylethanolamine-conjugated LC3 on the phagophore membrane. However, the detailed molecular mechanisms associated with the function of RavZ have hitherto remained unclear. Here, we report on the biochemical characteristics of the RavZ-LC3 interaction, the solution structure of the 1:2 complex between RavZ and LC3, and crystal structures of RavZ showing different conformations of the active site loop without LC3. Based on our biochemical, structural, and cell-based analyses of RavZ and LC3, both distant flexible N- and C-terminal regions containing LC3-interacting region (LIR) motifs are important for substrate recognition. These results suggest a novel mechanism of RavZ action on the phagophore membrane and lay the groundwork for understanding how bacterial pathogens can survive autophagy.     

Sulfonamide-based 4-anilinoquinoline derivatives as novel dual Aurora kinase (AURKA/B) inhibitors: Synthesis,biological evaluation and in silico insights

Aurora kinases (AURKs) were identified as promising druggable targets for targeted cancer therapy. Aiming at the development of novel chemotype of dual AURKA/B inhibitors, herein we report the design and synthesis of three series of 4-anilinoquinoline derivatives bearing a sulfonamide moiety (5a-d, 9a-d and 11a-d). The % inhibition of AURKA/B was determined for all target quinolines, then compounds showed more than 50% inhibition on either of the enzymes, were evaluated further for their IC₅₀ on the corresponding enzyme. In particular, compound 9d displayed potent AURKA/B inhibitory activities with IC₅₀ of 0.93 and 0.09 µM, respectively. Also, 9d emerged as the most efficient anti-proliferative analogue in the US-NCI anticancer assay toward the NCI 60 cell lines panel, with broad spectrum activity against different cell lines from diverse cancer subpanels. Docking studies, confirmed that, the sulfonamide SO₂ oxygen was involved in a hydrogen bond with Lys162 and Lys122 in AURKA and AURKB, respectively, whereas, the sulfonamide NH could catch hydrogen bond interaction with the surrounding amino acid residues Lys141, Glu260, and Asn261 in AURKA and Lys101, Glu177, and Asp234 in AURKB. Furthermore, N1 nitrogen of the quinoline scaffold formed an essential hydrogen bond with the hinge region key amino acids Ala213 and Ala173 in AURKA and AURKB, respectively.     

FLCN,a novel autophagy component,interacts with GABARAP and is regulated by ULK1 phosphorylation

Birt-Hogg-Dubé (BHD) syndrome is a rare autosomal dominant condition caused by mutations in the FLCN gene and characterized by benign hair follicle tumors, pneumothorax, and renal cancer. Folliculin (FLCN), the protein product of the FLCN gene, is a poorly characterized tumor suppressor protein, currently linked to multiple cellular pathways. Autophagy maintains cellular homeostasis by removing damaged organelles and macromolecules. Although the autophagy kinase ULK1 drives autophagy, the underlying mechanisms are still being unraveled and few ULK1 substrates have been identified to date. Here, we identify that loss of FLCN moderately impairs basal autophagic flux, while re-expression of FLCN rescues autophagy. We reveal that the FLCN complex is regulated by ULK1 and elucidate 3 novel phosphorylation sites (Ser406, Ser537, and Ser542) within FLCN, which are induced by ULK1 overexpression. In addition, our findings demonstrate that FLCN interacts with a second integral component of the autophagy machinery, GABA(A) receptor-associated protein (GABARAP). The FLCN-GABARAP association is modulated by the presence of either folliculin-interacting protein (FNIP)-1 or FNIP2 and further regulated by ULK1. As observed by elevation of GABARAP, sequestome 1 (SQSTM1) and microtubule-associated protein 1 light chain 3 (MAP1LC3B) in chromophobe and clear cell tumors from a BHD patient, we found that autophagy is impaired in BHD-associated renal tumors. Consequently, this work reveals a novel facet of autophagy regulation by ULK1 and substantially contributes to our understanding of FLCN function by linking it directly to autophagy through GABARAP and ULK1.     

FLCN,a novel autophagy component,interacts with GABARAP and is regulated by ULK1 phosphorylation

Birt-Hogg-Dubé (BHD) syndrome is a rare autosomal dominant condition caused by mutations in the FLCN gene and characterized by benign hair follicle tumors, pneumothorax, and renal cancer. Folliculin (FLCN), the protein product of the FLCN gene, is a poorly characterized tumor suppressor protein, currently linked to multiple cellular pathways. Autophagy maintains cellular homeostasis by removing damaged organelles and macromolecules. Although the autophagy kinase ULK1 drives autophagy, the underlying mechanisms are still being unraveled and few ULK1 substrates have been identified to date. Here, we identify that loss of FLCN moderately impairs basal autophagic flux, while re-expression of FLCN rescues autophagy. We reveal that the FLCN complex is regulated by ULK1 and elucidate 3 novel phosphorylation sites (Ser406, Ser537, and Ser542) within FLCN, which are induced by ULK1 overexpression. In addition, our findings demonstrate that FLCN interacts with a second integral component of the autophagy machinery, GABA(A) receptor-associated protein (GABARAP). The FLCN-GABARAP association is modulated by the presence of either folliculin-interacting protein (FNIP)-1 or FNIP2 and further regulated by ULK1. As observed by elevation of GABARAP, sequestome 1 (SQSTM1) and microtubule-associated protein 1 light chain 3 (MAP1LC3B) in chromophobe and clear cell tumors from a BHD patient, we found that autophagy is impaired in BHD-associated renal tumors. Consequently, this work reveals a novel facet of autophagy regulation by ULK1 and substantially contributes to our understanding of FLCN function by linking it directly to autophagy through GABARAP and ULK1.     

A novel BLyS antagonist peptide designed based on the 3-D complex structure of BCMA and BLyS

B lymphocyte stimulator (BLyS) is a member of tumor necrosis factor (TNF) family. Because of its roles in autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and Sjogren syndrome (SS), BLyS antagonists have been tested to treat SLE- and RA-like symptoms in mice and obtained optimistic results. So far, reported BLyS antagonists were mostly decoyed BLyS receptors or anti-BLyS antibodies. In this study, a novel BLyS antagonist peptide, PT, was designed based on the modeling 3-D complex structure of BCMA and BLyS. The interaction mode of PT with BLyS was analyzed theoretically. The results of competitive ELISA demonstrated that PT could inhibit the binding of BCMA-Fc and anti-BLyS antibody to BLyS in vitro. In addition, PT could partly block the proliferating activity of BLyS on mice splenocytes. The BLyS antagonizing activity of PT was significant (p<0.05). This study highlights the possibility of using BLyS antagonist peptide to neutralize BLyS activity. Further optimization of PT with computer-guided molecular design method to enhance its biopotency may be useful in developing new BLyS antagonists to treat BLyS-related autoimmune diseases.     

Optical isomers of a leukotriene B4 antagonist have differential effects on granulocyte diapedesis in the guinea pig dermis.

Leukotriene B4 (LTB4) is a proinflammatory product of arachidonic acid metabolism that has been implicated in a number of inflammatory diseases. When injected intradermally into the guinea pig, LTB4 has been shown to elicit a dose-dependent infiltration of granulocytes as assessed by the level of the neutrophil marker enzyme myeloperoxidase. SC-41930 [7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)propoxy]-3,4-dihydro-8- propyl-2H-1-benzopyran-2-carboxylic acid] is a potent LTB4 receptor antagonist. When compounds were coadministered along with LTB4 (35 ng) into the dermal site, racemic SC-41930, (+)-SC-41930, and (-)-SC-41930 each inhibited granulocyte accumulation with ED50 values of 340 +/- 30, 98 +/- 5.7, and 1000 +/- 142 ng, respectively; when given intravenously inhibited with ED50 values of 0.5 +/- 0.06, 0.3 +/- 0.04, and 1.4 +/- 0.19 mg/kg, respectively; and when given intragastrically inhibited with ED50 values of 1.7 +/- 0.20, 1.4 +/- 0.23, and 3.0 +/- 0.41 mg/kg, respectively.     

Isotyping of component C4 of human complement using differences in the functional activity of isotypes C4A and C4B

The difference in the functional activity of the isotypes A and B of component C4 of human complement was used to determine their ratio and to detect the inherited deficiency of the isotypes. ELISA methods were developed for the quantitative assay of component C4 (conventional sandwich method) and its functional activity. When determining the functional activity, the classic pathway of the complement and therefore of component C4 was activated on activators sorbed on ELISA microplates: immunoglobulin IgG3 or liposaccharide of theShigella sonnei cell walls, which activates the complement by binding component C1. The nascent fragment C4b is covalently bound to the target activator; C4Ab binds better to the target protein (immunoglobulin), and C4Bb to the target carbohydrate (liposaccharide). Therefore, when immunoglobulin is a target activator, isotype C4A is bound and determined; and when the complement is activated with liposaccharide, isotype C4B is determined. The radio of the activities determined by the two methods indicates the deficiency in the individual isotypes of component C4 or its absence. The rabbit polyclonal monospecific antibodies against the human component C4 and the conjugates of these antibodies with horseradish peroxidase were used in the methods described.