Biomedical subjects

Friedrich Lottspeich

Publications and source records attributed to Friedrich Lottspeich.

At least 37 records · Page 2Linked to original sources

PubMed2004

The 2.0 A crystal structure and substrate specificity of the KDEL-tailed cysteine endopeptidase functioning in programmed cell death of Ricinus communis endosperm.

In the senescing endosperm of germinating castor bean (Ricinus communis) a special organelle (the ricinosome) releases a papain-type cysteine endopeptidase (CysEP) during the final stages of cellular disintegration. Protein cleavage sites for the Ricinus CysEP were determined with fluorogenic peptides (Abz-Xaa-Arg-/-Gln-Gln-Tyr(NO2)-Asp). The highest kcat/Km values were obtained with neutral amino acid residues with large aliphatic and non-polar (Leu, Val, Ile, Met) or aromatic (Phe, Tyr, Trp) side-chains. A second series (Abz-Leu-Xaa-/Gln-Pro-Tyr(NO2)-Asp) was evaluated. Based on these results, the covalent binding inhibitor H-D-Val-Leu-Lys-chloromethylketone (CMK) was chosen as substrate analogue for replacement in the catalytic site. Unusually, CysEP cleaved beta-casein N and C-terminal to the amino acid proline. CysEP was crystallized, its structure was solved by molecular replacement at 2.0 A resolution and refined to a R-factor of 18.1% (Rfree=22.6%). The polypeptide chain folds as in papain into two domains divided by the active site cleft, an elongated surface depression harboring the active site. The non-primed specificity subsites of the proteinase are clearly defined by the H-D-Val-Leu-Lys-CMK-inhibitor covalently bound to the active site. The absence of the occluding loop, which blocks the active site of exopeptidases at the C-terminal side of the scissile bond, identifies CysEP as an endopeptidase. The more open pocket of the Ricinus CysEP correlates with the extended variety of substrate amino acid residues accommodated by this enzyme, including even proline at the P1 and P1' positions. This may allow the enzyme to attack a greater variety of proteins during programmed cell death.

Amino Acid Sequence↗

PubMed2004

Determination of the stoichiometry of protein complexes using liquid chromatography with fluorescence and mass spectrometric detection of fluorescently labeled proteolytic peptides.

A method for the determination of the stoichiometry of protein complexes has been developed, which is based on proteolytic digestion of the complex, labeling with a fluorescent reagent, specific for amino or sulfhydryl groups, and separation by liquid chromatography with fluorescence and mass spectrometric detection. The intensity of the fluorescence signal of the labeled peptides resulting from different proteins is directly proportional to the stoichiometry of these proteins in the complex. The performance of the method was evaluated with standard peptides and proteins to ensure that accurate molar ratios can be obtained from the fluorescence chromatogram. Standard deviations of the measured molar ratio from the expected molar ratio were below 10% for both peptides and proteins. The method was finally employed for the determination of the stoichiometry of the 1:1 complex of sFc gamma RIII and hFc1. Using the described methodology, a stoichiometry of 1:1.1 was measured, which agrees well with a 1:1 complex.

Angiotensins↗

PubMed2004

Functional interaction in establishment of ribosomal integrity between small subunit protein rpS6 and translational regulator rpL10/Grc5p.

Functional ribosomes synthesize proteins in all living cells and are composed of two labile associated subunits, which are made of rRNA and ribosomal proteins. The rRNA of the small 40S subunit (SSU) of the functional eukaryotic 80S ribosome decodes the mRNA molecule and the large 60S subunit (LSU) rRNA catalyzes protein synthesis. Recent fine structure determinations of the ribosome renewed interest in the role of ribosomal proteins in modulation of the core ribosomal functions. RpL10/Grc5p is a component of the LSU and is a multifunctional translational regulator, operating in 60S subunit biogenesis, 60S subunit export and 60S subunit joining with the 40S subunit. Here, we report that rpL10/Grc5p functionally interacts with the nuclear export factor Nmd3p in modulation of the cellular polysome complement and with the small subunit protein rpS6 in subunit joining and differential protein expression.

Base Sequence↗

PubMed2004

Isolation and Characterization of an Outer Membrane Protein of Chlorobium tepidum.

A protein was isolated from membranes of the green sulfur bacterium Chlorobium tepidum. This protein was characterized by gel electrophoresis, gel filtration, analytical ultracentrifugation and amino acid sequencing. The molecular weight of the purified protein was shown to be 26 kDa by SDS-PAGE. HPLC gelfiltration, SDS-PAGE and analytical ultracentrifugation are consistent with the presence of a homogenous protein in the preparations. Amino acid analysis was obtained from the isolated protein after fragmentation with Lys-C, trypsin and cyanogen bromide. The cleavage pattern resulting from these treatments combined with Edman sequencing yield a sequence allowing the identification of an integral membrane agglutinin in Chl. tepidum.

Chlorobium tepidum↗

PubMed2003

An exosome-like complex in Sulfolobus solfataricus.

We present the first experimental evidence for the existence of an exosome-like protein complex in Archaea. In Eukarya, the exosome is essential for many pathways of RNA processing and degradation. Co-immunoprecipitation with antibodies directed against the previously predicted Sulfolobus solfataricus orthologue of the exosome subunit ribosomal-RNA-processing protein 41 (Rrp41) led to the purification of a 250-kDa protein complex from S. solfataricus. Approximately half of the complex cosediments with ribosomal subunits. It comprises four previously predicted orthologues of the core exosome subunits from yeast (Rrp41, Rrp42, Rrp4 and Csl4 (cepl synthetic lethality 4; an RNA-binding protein and exosome sub-unit)), whereas other predicted subunits were not found. Surprisingly, the archaeal homologue of the bacterial DNA primase DnaG was tightly associated with the complex. This suggests an RNA-related function for the archaeal DnaG-like proteins. Comparison of experimental data from different organisms shows that the minimal core of the exosome consists of at least one phosphate-dependent ribonuclease PH homologue, and of Rrp4 and Csl4. Such a protein complex was probably present in the last common ancestor of Archaea and Eukarya.

Macromolecular Substances↗

PubMed2003

Isolation, characterization and electron microscopic single particle analysis of the NADH:ubiquinone oxidoreductase (complex I) from the hyperthermophilic eubacterium Aquifex aeolicus.

The proton-translocating NADH:ubiquinone oxidoreductase (complex I) has been purified from Aquifex aeolicus, a hyperthermophilic eubacterium of known genome sequence. The purified detergent solubilized enzyme is highly active above 50 degrees C. The specific activity for electron transfer from NADH to decylubiquinone is 29 U/mg at 80 degrees C. The A. aeolicus complex I is completely sensitive to rotenone and 2-n-decyl-quinazoline-4-yl-amine. SDS polyacrylamide gel electrophoresis shows that it may contain up to 14 subunits. N-terminal amino acid sequencing of the bands indicates the presence of a stable subcomplex, which is composed of subunits E, F, and G. The isolated complex is highly stable and active in a temperature range from 50 to 90 degrees C, with a half-life of about 10 h at 80 degrees C. The activity shows a linear Arrhenius plot at 50-85 degrees C with an activation energy at 31.92 J/mol K. Single particle electron microscopy shows that the A. aeolicus complex I has the typical L-shape. However, visual inspection of averaged images reveals many more details in the external arm of the complex than has been observed for complex I from other sources. In addition, the angle (90 degrees ) between the cytoplasmic peripheral arm and the membrane intrinsic arm of the complex appears to be invariant.

Bacteria↗

PubMed2003

Taming of a poison: biosynthesis of the NiFe-hydrogenase cyanide ligands.

NiFe-hydrogenases have an Ni-Fe site in which the iron has one CO and two CN groups as ligands. Synthesis of the CN ligands requires the activity of two hydrogenase maturation proteins: HypF and HypE. HypF is a carbamoyltransferase that transfers the carbamoyl moiety of carbamoyladenylate to the COOH-terminal cysteine of HypE and thus forms an enzyme-thiocarbamate. HypE dehydrates the S-carbamoyl moiety in an adenosine triphosphate-dependent process to yield the enzyme thiocyanate. Chemical model reactions corroborate the feasibility of this unprecedented biosynthetic route and show that thiocyanates can donate CN to iron. This finding underscores a striking parallel between biochemistry and organometallic chemistry in the formation of an iron-cyano complex.

Adenosine Monophosphate↗

PubMed2003

Analysis of the quorum-sensing regulon of the opportunistic pathogen Burkholderia cepacia H111 by proteomics.

Burkholderia cepacia H111, an important pathogen for persons suffering from cystic fibrosis, employs a quorum-sensing (QS) system, cep, to control expression of virulence factors as well as the formation of biofilms. The QS system is thought to ensure that pathogenic traits are only expressed when the bacterial population density is high enough to overwhelm the host before it is able to mount an efficient response. In this study, we compared the protein pattern of the intracellular, extracellular, and surface protein fractions of an AHL-deficient cepI mutant with the one of the parent strain H111 by means of two-dimensional gel electrophoresis (2-DE). Our analysis showed that 55 proteins out of 985 detected spots were differentially expressed; these are expected to represent QS-controlled gene products. Addition of the respective signal molecules to the growth medium of the cep mutant fully restored the wild-type protein expression profile. In total about 5% of the B. cepacia proteome was downregulated and 1% upregulated in the cepI mutant, indicating that quorum sensing represents a global regulatory system. Nineteen proteins were identified with high confidence by N-terminal sequence analysis.

Burkholderia cepacia↗

PubMed2003

Identification of markers for the selection of patients undergoing renal cell carcinoma-specific immunotherapy.

Renal cell carcinoma (RCC) represents the most common malignant tumor in the kidney and is resistant to conventional therapies. The diagnosis of RCC is often delayed leading to progression and metastatic spread of the disease. Thus, validated markers for the early detection of the disease as well as selection of patients undergoing specific therapy is urgently needed. Using treatment with the monoclonal antibody (mAb) G250 as a model, proteome-based strategies were implemented for the identification of markers which may allow the discrimination between responders and nonresponders prior to application of G250-mediated immunotherapy. Flow cytometry revealed G250 surface expression in approximately 40% of RCC cell lines, but not in the normal kidney epithelium cell lines. G250 expression levels significantly varied thereby distinguishing between low, medium and high G250 expressing cell lines. Comparisons of two-dimensional gel electrophoresis expression profiles of untreated RCC cell lines versus RCC cell lines treated with a mAb directed against G250 and the characterization of differentially expressed proteins by mass spectrometry and/or Edman sequencing led to the identification of proteins such as chaperones, antigen processing components, transporters, metabolic enzymes, cytoskeletal proteins and unknown proteins. Moreover, some of these differentially expressed proteins matched with immunoreactive proteins previously identified by proteome analysis combined with immunoblotting using sera from healthy donors and RCC patients, a technique called PROTEOMEX. Immunohistochemical analysis of a panel of surgically removed RCC lesions and corresponding normal kidney epithelium confirmed the heterogeneous expression pattern found by proteome-based technologies. In conclusion, conventional proteome analysis as well as PROTEOMEX could be successfully employed for the identification of markers which may allow the selection of patients prior to specific immunotherapy.

Antibodies, Monoclonal↗

PubMed2003

Human IgG1/IgG3 cryoglobulin suggesting lack of allelic exclusion.

Immunoglobulins undergoing cold-dependent precipitation are known as cryoglobulins. A type I cryoglobulin after Brouet et al. from serum of a patient with severe cutaneous vasculitis and membranoproliferative glomerulonephritis was purified by reversible temperature-dependent precipitation and analyzed using FPLC, Western blotting and peptide sequencing. The isolated cryoglobulin consisted of a single complex of a molecular weight of above 210kDa observed under non-reducing conditions in SDS-polyacrylamide gel electrophoresis (PAGE). Under reducing conditions, this complex resolved into three bands, two of which were reminiscent of Ig heavy (HC) chains and one of Ig-light chains (LC). The FPLC-purified type I cryoglobulin showed reversible precipitation analyzed by spectrophotometry. Delineation of the peptides involved in complex formation by immunoblot analysis and peptide sequencing revealed IgG3-V(H)4/Igkappa-VkappaIII/JkappaII and IgG1/V(H)3 molecules with evidence of somatic mutation. Coomassie blue-staining suggested that molar amounts of the IgG3-heavy chain were much higher than that of the IgG1-heavy chain. Treatment with SDS and boiling did not disrupt the unusually high molecular weight Ig complex. Pre-treatment of the cryoglobulin in 6M guadinium hydrochloride followed by gel filtration chromatography suggested covalent association of the IgG3, IgG1 and Igkappa molecules. Therefore, it might be that the cryoglobulin was produced by a single plasma B cell clone which passed immunological check-points in terms of B cell selection in the bone marrow in the absence of allelic exclusion, class switching and affinity maturation by somatic mutation.

Aged↗

PubMed2003

Identification of teleost Thy-1 and association with the microdomain/lipid raft reggie proteins in regenerating CNS axons.

During regeneration, retinal ganglion cell axons in fish upregulate a cell surface protein that is recognized by the monoclonal antibody (mAB) M802. M802 antigen appeared to be linked to the intracellular, membrane-associated lipid raft/microdomain proteins reggie-1 and reggie-2 that were previously shown to be reexpressed in axon-regenerating neurons [Development 124 (1997), 577]. Here, we report the isolation of the M802 antigen and its identification as the teleost homolog of mammalian Thy-1. Fish Thy-1 is detected in the same detergent-insoluble lipid raft fractions from a fibroblast cell line and from axon regenerating retinae as reggie-1 and 2. Importantly, mAB M802 coimmunoprecipitates reggie-1 and 2 from this lipid raft fraction, implying that fish Thy-1 and reggies interact. This correlates with their colocalization in growing cell processes after M802 antigen/Thy-1 activation with mAB M802. These findings suggest a role of clustered M802 antigen/Thy-1 in reggie raft microdomains for cell growth and axon regeneration.

Animals↗

PubMed2003

Identification of an anion-specific channel in the cell wall of the Gram-positive bacterium Corynebacterium glutamicum.

A cation-selective channel (porin), designated PorA, facilitates the passage of hydrophilic solutes across the cell wall of the mycolic acid-containing actinomycete Corynebacterium glutamicum. Biochemical and electrophysiological investigations of the cell wall of the mutant strain revealed the presence of an alternative channel-forming protein. This porin was purified to homogeneity and studied in lipid bilayer membranes. It forms small anion-selective channels with a diameter of about 1.4 nm and an average single-channel conductance of about 700 pS in 1 M KCl. The PorBCglut channel could be blocked by citrate in a dose-dependent manner. This result was in agreement with growth experiments in citrate as sole carbon source where growth in citrate was impaired as compared with growth in other carbon sources. The PorBCglut protein was partially sequenced and based on the resulting amino acid sequence of the corresponding gene, which was designated as porB, was identified as an unannotated 381 bp long open reading frame (ORF) in the published genome sequence of C. glutamicum ATCC13032. PorBCglut contains 126 amino acids with an N-terminal extension of 27 amino acids. One hundred and thirty-eight base pairs downstream of porB, we found an ORF that codes for a protein with about 30% identity to PorBCglut, which was named PorCCglut. The arrangement of porB and porC on the chromosome suggested that both genes belong to the same cluster. RT-PCR from overlapping regions between genes from wild-type C. glutamicum ATCC 13032 and its ATCC 13032DeltaporA mutant demonstrated that this is the case and that porB and porC are cotranscribed. The gene products PorBCglut and PorCCglut represent obviously other permeability pathways for the transport of hydrophilic compounds through the cell wall of C. glutamicum.

Amino Acid Sequence↗

PubMed2003

(R,S)-Reticuline 7-O-methyltransferase and (R,S)-norcoclaurine 6-O-methyltransferase of Papaver somniferum - cDNA cloning and characterization of methyl transfer enzymes of alkaloid biosynthesis in opium poppy.

S-Adenosyl-L-methionine:(R,S)-reticuline 7-O-methyltransferase converts reticuline to laudanine in tetrahydrobenzylisoquinoline biosynthesis in the opium poppy Papaver somniferum. This enzyme activity has not yet been detected in plants. A proteomic analysis of P. somniferum latex identified a gel spot that contained a protein(s) whose partial amino acid sequences were homologous to those of plant O-methyltransferases. cDNA was amplified from P. somniferum RNA by reverse transcription PCR using primers based on these internal amino acid sequences. Recombinant protein was then expressed in Spodoptera frugiperda Sf9 cells in a baculovirus expression vector. Steady-state kinetic measurements with one heterologously expressed enzyme and mass spectrometric analysis of the enzymatic products suggested that this unusual enzyme is capable of carrying through sequential O-methylations on the isoquinoline and on the benzyl moiety of several substrates. The tetrahydrobenzylisoquinolines (R)-reticuline (4.2 sec(-1) mm(-1)), (S)-reticuline (4.5 sec(-1) mm(-1)), (R)-protosinomenine (1.7 sec(-1) mm(-1)), and (R,S)-isoorientaline (1.4 sec(-1) mm(-1)) as well as guaiacol (5.9 sec(-1) mm(-1)) and isovanillic acid (1.2 sec(-1) mm(-1)) are O-methylated by the enzyme with the ratio kcat/K m shown in parentheses. A P. somniferum cDNA encoding (R,S)-norcoclaurine 6-O-methyltransferase was similarly isolated and characterized. This enzyme was less permissive, methylating only (R,S)-norcoclaurine (7.4 sec(-1) mm(-1)), (R)-norprotosinomenine (4.1 sec(-1) mm(-1)), (S)-norprotosinomenine (4.0 sec(-1) mm(-1)) and (R,S)-isoorientaline (1.0 sec(-1) mm(-1)). A phylogenetic comparison of the amino acid sequences of these O-methyltransferases to those from 28 other plant species suggests that these enzymes group more closely to isoquinoline biosynthetic O-methyltransferases from Coptis japonica than to those from Thalictrum tuberosum that can O-methylate both alkaloid and phenylpropanoid substrates.

Alkaloids↗

PubMed2003

Lic16A of Clostridium thermocellum, a non-cellulosomal, highly complex endo-beta-1,3-glucanase bound to the outer cell surface.

Clostridium thermocellum produces one major beta-1,3-glucanase. Genomic DNA fragments containing the gene were cloned from two strains, DSM1237(T) (6848 bp) and F7 (9766 bp). Overlapping sequences were 99.9 % identical. The nucleotide sequences contained reading frames for a putative transposase, endo-beta-1,3-1,4-glucanase CelC, a putative transcription regulator of the LacI type, beta-1,3-glucanase Lic16A and a putative membrane protein. The licA genes of both strains encoded an identical protein of 1324 aa with a calculated molecular mass of 148 kDa. Lic16A is an unusually complex protein consisting of a leader peptide, a threefold repeat of an S-layer homologous module (SLH), an unknown module, a catalytic module of glycosyl hydrolase family 16 and a fourfold repeat of a carbohydrate-binding module of family CBM4a. The recombinant Lic16A protein was characterized as an endo-1,3(4)-beta-glucanase with a specific activity of 2680 and 340 U mg(-1) and a K(m) of 0.94 and 2.1 mg ml(-1) towards barley beta-glucan and laminarin, respectively. It was specific for beta-glucans containing beta-1,3-linkages with an optimum temperature of 70 degrees C at pH 6.0. The N-terminal SLH modules were cleaved from the protein as well in Escherichia coli as in C. thermocellum, but nevertheless bound tightly to the rest of the protein. Lic16A was located on the cell surface from which it could be purified after fractionated solubilization. Its inducible production allowed C. thermocellum to grow on beta-1,3- or beta-1,3-1,4-glucan.

Amino Acid Sequence↗

PubMed2003

Membrane-bound hydrogenase and sulfur reductase of the hyperthermophilic and acidophilic archaeon Acidianus ambivalens.

A sulfur reductase (SR) and a hydrogenase were purified from solubilized membrane fractions of anaerobically grown cells of the sulfur-dependent archaeon Acidianus ambivalens and the corresponding genes were sequenced. The SR reduced elemental sulfur with hydrogen as electron donor [45 U (mg protein)(-1)] in the presence of hydrogenase and either 2,3-dimethylnaphthoquinone (DMN) or cytochrome c in the enzyme assay. The SR could not be separated from the hydrogenase during purification without loss of activity, whereas the hydrogenase could be separated from the SR. The specific activity of the hydrogenase was 170 U (mg protein)(-1) with methyl viologen and 833 U (mg protein)(-1) with DMN as electron acceptors. Both holoenzymes showed molecular masses of 250 kDa. In SDS gels of active fractions, protein bands with apparent masses of 110 (SreA), 66 (HynL), 41 (HynS) and 29 kDa were present. Enriched hydrogenase fractions contained 14 micro mol Fe and 2 micromol Ni (g protein)(-1); in addition, 2.5 micromol Mo (g protein)(-1) was found in the membrane fraction. Two overlapping genomic cosmid clones were sequenced, encoding a five-gene SR cluster (sre) including the 110 kDa subunit gene (sreA), and a 12-gene hydrogenase cluster (hyn) including the large and small subunit genes and genes encoding proteins required for the maturation of NiFe hydrogenases. A phylogenetic analysis of the SR amino acid sequence revealed that the protein belonged to the DMSO reductase family of molybdoenzymes and that the family showed a novel clustering. A model of sulfur respiration in Acidianus developed from the biochemical results and the data of the amino acid sequence comparisons is discussed.

Acidianus↗

PubMed2003

Inactivation of the selB gene in Methanococcus maripaludis: effect on synthesis of selenoproteins and their sulfur-containing homologs.

The genome of Methanococcus maripaludis harbors genes for at least six selenocysteine-containing proteins and also for homologs that contain a cysteine codon in the position of the UGA selenocysteine codon. To investigate the synthesis and function of both the Se and the S forms, a mutant with an inactivated selB gene was constructed and analyzed. The mutant was unable to synthesize any of the selenoproteins, thus proving that the gene product is the archaeal translation factor (aSelB) specialized for selenocysteine insertion. The wild-type form of M. maripaludis repressed the synthesis of the S forms of selenoproteins, i.e., the selenium-independent alternative system, in selenium-enriched medium, but the mutant did not. We concluded that free selenium is not involved in regulation but rather a successional compound such as selenocysteyl-tRNA or some selenoprotein. Apart from the S forms, several enzymes from the general methanogenic route were affected by selenium supplementation of the wild type or by the selB mutation. Although the growth of M. maripaludis on H(2)/CO(2) is only marginally affected by the selB lesion, the gene is indispensable for growth on formate because M. maripaludis possesses only a selenocysteine-containing formate dehydrogenase.

Amino Acid Sequence↗

PubMed2002

Novel basic-region helix-loop-helix transcription factor (AnBH1) of Aspergillus nidulans counteracts the CCAAT-binding complex AnCF in the promoter of a penicillin biosynthesis gene.

Cis-acting CCAAT elements are found frequently in eukaryotic promoter regions. Many of the genes containing such elements in their promoters are regulated by a conserved multimeric CCAAT-binding complex. In the fungus Emericella (Aspergillus) nidulans, this complex was designated AnCF (A.nidulans CCAAT-binding factor). AnCF regulates several genes, including the penicillin biosynthesis genes ipnA and aatA. Since it is estimated that the CCAAT-binding complex regulates more than 200 genes, an important question concerns the regulation mechanism that allows so many genes to be regulated by a single complex in a gene-specific manner. One of the answers to this question appears to lie in the interaction of AnCF with other transcription factors. Here, a novel transcription factor designated AnBH1 was isolated. The corresponding anbH1 gene was cloned and found to be located on chromosome IV. The deduced AnBH1 protein belongs to the family of basic-region helix-loop-helix (bHLH) transcription factors. AnBH1 binds in vitro as a homodimer to an, not previously described, asymmetric E-box within the aatA promoter that overlaps with the AnCF-binding site. This is the first report demonstrating that the CCAAT-binding complex and a bHLH transcription factor bind to overlapping sites. Since deletion of anbH1 appears to be lethal, the anbH1 gene was replaced by a regulatable alcAp-anbH1 gene fusion. The analysis of aatAp-lacZ expression in such a strain indicated that AnBH1 acts as a repressor of aatA gene expression and therefore counteracts the positive action of AnCF.

Amino Acid Sequence↗

PubMed2002

Nowa, a novel protein with minicollagen Cys-rich domains, is involved in nematocyst formation in Hydra.

The novel protein Nowa was identified in nematocysts, explosive organelles of Hydra, jellyfish, corals and other CNIDARIA: Biogenesis of these organelles is complex and involves assembly of proteins inside a post-Golgi vesicle to form a double-layered capsule with a long tubule. Nowa is the major component of the outer wall, which is formed very early in morphogenesis. The high molecular weight glycoprotein has a modular structure with an N-terminal sperm coating glycoprotein domain, a central C-type lectin-like domain, and an eightfold repeated cysteine-rich domain at the C-terminus. Interestingly, the cysteine-rich domains are homologous to the cysteine-rich domains of minicollagens. We have previously shown that the cysteines of these minicollagen cysteine-rich domains undergo an isomerization process from intra- to intermolecular disulfide bonds, which mediates the crosslinking of minicollagens to networks in the inner wall of the capsule. The minicollagen cysteine-rich domains present in both proteins provide a potential link between Nowa in the outer wall and minicollagens in the inner wall. We propose a model for nematocyst formation that integrates cytoskeleton rearrangements around the post-Golgi vesicle and protein assembly inside the vesicle to generate a complex structure that is stabilized by intermolecular disulfide bonds.

Amino Acid Sequence↗