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Query: UNIPROT:P06889 (Mol)
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Two monoclonal antibodies, designated 1F8 (IgG1) and 5B10 (IgG1), have been produced in mice against native human type III collagen. These antibodies were highly type and species specific, recognizing the triple helical domain of type III as tested by ELISA. Immunofluorescence studies using each of these antibodies resulted in a fibrous staining pattern in human skin dermis. Immunogold electron microscopy resulted in a periodic distribution of gold particulates along banded collagen fibrils. Assuming that the total contour length of pepsin digested type III collagen is 300 nm, measurements of antibody-antigen complexes visualized by rotary shadowing revealed that each antibody bound at the same two sites: one approximately at the middle of the helix (153 nm from the N-terminus), the other at a site one-quarter the triple helical length from the N-terminus (75 nm). That the one-quarter binding site was closest to the N-terminus was determined by antibody incubation following tadpole collagenase treatment, which results in a larger, N-terminus containing fragment (binding antibody) and a smaller C-terminus containing fragment (not binding antibody). Located at each antibody binding epitope is a sequence of 10 amino acids: Gly-Ala-Hyp-Gly-Leu-Arg-Gly-Gly-Ala-Gly. Renatured cyanogen bromide-cleaved(CB)-peptides, CB4 and CB8, containing these repeated sequences reacted with each antibody, whereas other renatured type III CB-peptides were unreactive as determined by Western blotting analysis and ELISA. This was further confirmed by inhibition tests using a 10 residue synthetic peptide of identical sequence, which yielded 20-30% inhibition of antibody binding to native type III collagen at 4 degrees C. However, no inhibition was noted at higher temperature. These results indicate that both monoclonal antibodies recognize a specific helical conformation of 10 or slightly fewer residues in the three identical polypeptide chains comprising type III collagen.
Mol Immunol 1992 Jun
PMID:Repeated helical epitopes of defined amino acid sequence in human type III collagen identified by monoclonal antibodies. 137 14

Cathepsin D was purified from bovine liver by a method using two pepstatin A affinity columns. The eluted protein was combined with pepstatin A and the complex crystallized from 15% polyethylene glycol 8000 at pH 5.9. The crystals diffract to a resolution of 3.0 A and have space group P2(1)2(1)2(1) with unit cell dimensions a = 74.8 A, b = 76.0 A, c = 157.7 A. There are two molecules in the asymmetric unit. The structure was solved by molecular replacement using a pepsin search model and both molecules showed clearly interpretable density in the position expected for pepstatin A in a preliminary difference map. The refined model has r.m.s. deviations from ideal bond lengths and angles of 0.014 A and 3.2 degrees, respectively, and a crystallographic R factor of 17%.
J Mol Biol 1992 Oct 20
PMID:Crystallization and initial crystallographic results for pepstatin A inhibited bovine cathepsin D. 143

An IgM(kappa) immunoglobulin from a patient (Pot) with Waldenstrom's macroglobulinemia was hydrolyzed with pepsin to release a fragment consisting of the 'variable' (V) domains of the light and heavy chains plus eight residue 'tails' from the 'constant' (C) domains. The crystal structure of this fragment was determined at 2.3 A resolution by molecular replacement and crystallographic refinement methods. When examined separately, the light chain component closely resembles another human kappa chain (Rei) in both the beta-pleated sheet regions and the 'hypervariable' loops. The conserved pleated sheets in the heavy chain are similar to those in the human Kol IgG1 protein, but the third hypervariable loop in particular is different from that in any immunoglobulin structure described to date. As in the Kol protein, this loop blocks the access to any internal active site along the light-heavy chain interface. Unlike the Kol protein, however, the loop does not protrude beyond the boundaries of a conventional antigen combining site. Instead, it forms a very compact structure, which fills almost all residual space between the domains. This is an example of one dominant complementarity-determining region (CDR) essentially negating the diversity possible with five other CDRs in the two chains. Ordered water molecules are associated with light chain constituents along the interface, but not with CDR3 of the heavy chain. In screening exercises the Pot IgM failed to bind a wide variety of peptides. Together, the results suggest that ligand binding can only occur on external surfaces of the protein. These surfaces carry a limited number of side chains usually assigned to CDRs in more typical antibodies.
J Mol Biol 1992 Nov 05
PMID:Three-dimensional structure of an Fv from a human IgM immunoglobulin. 144 81

The partial amino acid sequences of the gamma chains of the bovine IgG2a(A1) and IgG2a(A2) allotypes were determined. Sequence differences were found in the CH1 domain, the hinge region, and the CH3 domain. The hinge regions displayed only 71.4% similarity and all of the differences were of a radical nature. The A2 hinge has isoleucine instead of serine at 229, histidine for asparagine at 235, proline for histidine at 238, and cysteine instead of proline in position 234; the latter has the potential for forming an additional interheavy chain disulphide bridge. The occurrence of such a bridge could explain the presence of a pepsin fragment consisting of the hinge region and the Fc. A corresponding fragment is not obtained with the A1 allotype. Both allotypes have a shortened hinge region and a truncated CH2 domain. This feature is characteristic of all reported sequences of IgG2 proteins but not IgG1 in cattle and the goat. This structural feature may be important in subclass-specific recognition by Fc gamma receptors in ruminants. A surprising discovery was the occurrence of five substitutions in the CH3 domain of the IgG2a(A2) in comparison with the A1, which are shared with the CH3 of IgG1. These permit the occurrence of isoallotypic determinants and can explain the difficulty encountered in preparing A2-specific antisera during which adsorption with IgG1 is a routine procedure. The primary sequence data we report confirm the presence of major structural differences between the A allotypes of cattle that was suggested by previous work. The sequence of the A1 allotype most closely agrees with the two IgG2 sequences deduced from their nucleotide sequences whereas the sequence differences in the hinge and C-terminal CH3 make IgG2a(A2) unique. The structural differences between allotypes could have major consequences for such biological activities as phagocytosis, transepithelial transport, lymphocyte and complement activation.
Mol Immunol 1992 Sep
PMID:The heterogeneity of bovine IgG2--V. Differences in the primary structure of bovine IgG2 allotypes. 149 1

Proteinase A from Aspergillus niger var. macrosporus is a non-pepsin-type acid proteinase distinctly different in various properties from the family of pepsin-type aspartic proteinases, and so far it remains unknown which residues participate in the catalysis of the enzyme and how the mechanism operates. The acid proteinase A was crystallized from an ammonium sulfate solution by the hanging-drop vapor diffusion method. The space group of the crystals was P2(1)2(1)2(1) with unit cell dimensions of a = 54.7 A, b = 70.4 A and c = 38.0 A. On the assumption that there is one enzyme molecule in the asymmetric unit, the calculated ratio of volume to unit protein mass (Vm) was 1.64 A3 per dalton. Diffraction data were collected up to a resolution higher than 1.5 A, using the Weissenberg camera for macromolecular crystallography with synchrotron radiation. The crystal of proteinase A is, therefore, suitable for the structural analysis with a high resolution.
J Mol Biol 1992 Jan 05
PMID:Crystallization and preliminary X-ray investigation of proteinase A, a non-pepsin-type acid proteinase from Aspergillus niger var. macrosporus. 173 Oct 82

T protein is a trypsin- and pepsin-resistant molecule on the surface of group A streptococci used as a serological tool to differentiate streptococci of this group. The purpose of this study was to determine the relatedness among the T protein genes of the 25 known T serotypes. DNA probes were constructed which represented various regions of the structural gene for the T6 protein, tee6. The probes were assayed for their ability to hybridize HindIII digests of chromosomal DNA from the 25 different T serotypes. Probe pTEE6.3, coding for the entire T6 protein, and pTEE6(1-299), coding for the amino-terminal half of T6, displayed the highest amount of homology, each binding to 10 of 25 T serotypes. Probes coding for sequences in the carboxy-terminal half of T6 showed considerably less homology among T serotypes with one probe hybridizing with only three out of 25. A synthetic oligonucleotide coding for the carboxy-terminal hydrophobic domain of T6, an area conserved to some degree among several bacterial surface proteins, showed homology with only seven out of 25 T serotypes. Hybridization with sequences outside the tee6 coding area provided additional information on the relatedness of certain sets of T serotypes according to restriction-fragment size heterogeneity. Clearly, there is considerable diversity among T-serotype genes. The data suggest that two or more families of structurally variant T proteins exist, which share only the property of proteolytic resistance and/or, perhaps, some biological function.
Mol Microbiol 1991 Dec
PMID:Genetic diversity among the T-protein genes of group A streptococci. 180 37

The isolation and characterization of an isotype-specific autoantibody-secreting hybridoma NET/2/3 from rats bearing the syngeneic tumour HSN is described. This rheumatoid factor of the IgM class recognizes an epitope within the hinge region of rat immunoglobulins of the IgG2b subclass which is destroyed by reduction of disulphide bonds. The specificity of NET/2/3, although not allotype-restricted, is highly isotype-restricted, as it does not bind to rat Ig other than IgG2b, nor does it react with the majority of mouse IgG, although some reactivity occurs with mouse IgG3. One remarkable feature of NET 2/3 is that it binds more strongly to F(ab')2 and Fab' fragments of rat IgG2b, obtained by digestion with pepsin, than to the whole molecule. This anti-isotype response is not peculiar to the HSN tumour model as NET/2/3-like antibodies have been found in the sera of rats immunized with various protein and cellular antigens. The possible biological role of this anti-isotype antibody is discussed.
Mol Immunol 1991 Jun
PMID:Isolation and characterization of a monoclonal rheumatoid factor specific for the hinge region of rat IgG2b. 186 82

Pleural fibrosis may complicate several types of non-exudative pleural injury. Although the pathogenesis of such lesions is poorly understood, it is conceivable that mesothelial cells may recruit fibroblasts to sites of pleural damage. In order to test this possibility, conditioned medium from cultured rat mesothelial cells was tested for chemoattractant activity towards RL-87 rat lung fibroblasts. For this purpose, rat pleural or pericardial mesothelial cells were maintained in vitro for 6 to 96 h. Conditioned medium from each source was obtained at defined culture times and tested for chemotactic activity in a 48-well microchemotaxis assembly. A progressive, time-dependent increase in fibroblast chemoattractant activity was detected in both pleural and pericardial mesothelial cell conditioned medium samples. This effect was maximal in 96-h cultures. Checkerboard analysis revealed that the conditioned medium was truly chemotactic for lung fibroblasts. Characterization of the chemoattractant demonstrated that it was a nondialyzable (greater than 16 kD), thermolabile (100 degrees C for 15 min), acid-stable (pH 2.5), trypsin-sensitive, and pepsin-sensitive protein. The chemotaxin was shown to be fibronectin, since activity was abolished, in a dose-dependent manner, by treatment with anti-rat fibronectin antiserum as well as by passage through a gelatin agarose affinity column. This product consisted of two bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis of apparent molecular masses 250 and 220 kD. The secretion of a mesothelial cell-derived fibroblast chemoattractant may play a role in the response of the pleura to injury and in the pathogenesis of pleural fibrosis.
Am J Respir Cell Mol Biol 1991 Sep
PMID:Mesothelial cells produce a chemoattractant for lung fibroblasts: role of fibronectin. 191 Aug 11

Two different collagens were isolated and characterized from the body walls of the vestimentiferan tube worm Riftia pachyptila and the annelid Alvinella pompejana, both living around hydrothermal vents at a depth of 2600 m. The acid-soluble cuticle collagens consisted of a long triple helix (2.4 microns for Alvinella, 1.5 microns for Riftia) terminating into a globular domain. Molecular masses of 2600 and 1700 kDa, respectively, were estimated from their dimensions. The two cuticle collagens were also quite different in amino acid composition, in agreement with their different supramolecular organizations within tissues. Interstitial collagens corresponding to cross-striated fibrils underneath the epidermal cells could be solubilized by digestion with pepsin and consisted of a single alpha-chain. They were similar in molecular mass (340 kDa) and length (280 nm) but differed in composition and banding patterns of segment-long-spacing fibrils. This implicates significant sequence differences also in comparison to fibril-forming vertebrate collagens, although all form typical quarter-staggered fibrils. The thermal stability of the worm collagens was, with one exception (interstitial collagen of Riftia), in the range of mammalian and bird collagens (37 to 46 degrees C), and thus distinctly above that of shallow sea water annelids. Yet, their 4-hydroxyproline contents were not directly correlated to this stability. About 20% of Riftia collagen alpha-chain sequence was elucidated by Edman degradation and showed typical Gly-X-Y repeats but only a limited homology (45 to 58% identity) to fibril-forming vertebrate collagens. A single triplet imperfection and the variable hydroxylation of proline in the X position were additional unique features. It suggests that this collagen represents an ancestral form of fibril-forming collagens not directly corresponding to an individual fibril-forming collagen type of vertebrates.
J Mol Biol 1991 Sep 05
PMID:Molecular characterization of cuticle and interstitial collagens from worms collected at deep sea hydrothermal vents. 192 Apr 5

The structure of calf chymosin (EC 3.4.23.3), the aspartic proteinase from the gastric mucosa, was solved using the technique of molecular replacement. We describe the use of different search models based on distantly related fungal aspartic proteinases and investigate the effect of using only structurally conserved regions. The structure has been refined to a crystallographic R-factor of 17% at 2.2 A resolution with an estimated co-ordinate error of 0.21 A. In all, 136 water molecules have been located of which eight are internal. The structure of chymosin resembles that of pepsin and other aspartic proteinases. However, there is a considerable rearrangement of the active-site "flap" and, in particular, Tyr75 (pepsin numbering), which forms part of the specificity pockets S1 and S1'. This is probably a consequence of crystal packing. Electrostatic interactions on the edge of the substrate binding cleft appear to account for the restricted proteolysis of the natural substrate kappa-casein by chymosin. The local environment of invariant residues is examined, showing that structural constraints and side-chain hydrogen bonding can play an important role in the conservation of particular amino acids.
J Mol Biol 1991 Oct 20
PMID:X-ray analyses of aspartic proteinases. IV. Structure and refinement at 2.2 A resolution of bovine chymosin. 194 52


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