Intracellular avian type X collagen in situ and determination of its thermal stability using a conformation-dependent monoclonal antibody

The thermal stability of the helical domain of intracellular and matrix-associated type X collagen was examined in situ within the hypertrophic region of embryonic chick vertebral cartilages. For this we employed indirect immunofluorescence histochemistry of unfixed tissue sections reacted at progressively higher temperatures (Linsenmayer et al., J cell biol 99 (1984) 1405) with a conformation-dependent monoclonal antibody (X-AC9) (Schmid & Linsenmayer, J cell biol 100 (1985) 598). The hypertrophic chondrocytes which had most recently initiated synthesis of type X did not immediately secrete it, but instead retained it intracellularly within cytoplasmic organelles. This allowed for clear visualization of the intracellular type X. Within the pool of intracellular type X collagen, the epitope recognized by the antibody was stable up to 55 degrees C, but was destroyed at 60 degrees C. This is 5-10 degrees C higher than the thermal stability of the epitope when the molecule is in neutral solution (as determined by competition ELISA). The matrix-associated type X collagen is stable at least to 65-67.5 degrees C. We conclude that in situ the stability of the collagen helix in its normal intracellular environment is considerably greater than might be predicted from measurements made on molecules in solution.     

Domain and basement membrane specificity of a monoclonal antibody against chicken type IV collagen

A monoclonal antibody, IV-IA8, generated against chicken type IV collagen has been characterized and shown to bind specifically to a conformational-dependent site within a major, triple helical domain of the type IV molecule. Immunohistochemical localization of the antigenic determinant with IV-IA8 revealed that the basement membranes of a variety of chick tissues were stained but that the basement membrane of the corneal epithelium showed little, if any, staining. Thus, basement membranes may differ in their content of type IV collagen, or in the way in which it is assembled. The specificity of the antibody was determined by inhibition ELISA using purified collagen types I-V and three purified molecular domains of chick type IV collagen ([F1]2F2, F3, and 7S) as inhibitors. Only unfractionated type IV collagen and the (F1)2F2 domain bound the antibody. Antibody binding was destroyed by thermal denaturation of the collagen, the loss occurring at a temperature similar to that at which previous optical rotatory dispersion studies had shown melting of the triple helical structure of (F1)2F2. Such domain-specific monoclonal antibodies should prove to be useful probes in studies involving immunological dissection of the type IV collagen molecule, its assembly within basement membranes, and changes in its distribution during normal development and in disease.     

Mutations in the alpha 2(IV) basement membrane collagen gene of Caenorhabditis elegans produce phenotypes of differing severities.

Type IV collagen forms a network that provides the major structural support of basement membranes. We have determined the nucleotide alterations and phenotypes of 17 mutant alleles of the Caenorhabditis elegans alpha 2(IV) collagen gene let-2. All 17 mutations are within the triple helical (Gly-X-Y) repeat domain of the molecule. Fifteen of the mutations are replacements of Gly-X-Y repeat glycines with aspartate, glutamate or arginine, and they cause a wide range of phenotypes. The mildest alleles are nearly wild-type at 15 and 20 degrees C but embryonic lethal at 25 degrees C, while the most severe allele is embryonic lethal at all three temperatures. Mutations resulting in severe phenotypes are generally located in areas of lower calculated thermal stability of the type IV collagen molecule. An alanine to threonine substitution at position X of a Gly-X-Y triplet immediately following an interruption results in a severe phenotype. This mutation is unusual because substitutions at positions X or Y have not generally been found to cause strong phenotypes in C. elegans or human collagens. An intron splice acceptor mutation causes a strict embryonic lethal phenotype, but does not completely abolish gene function. Pairs of independent mutations affect each of three glycines, indicating a non-random distribution of mutations in the molecule. It is suggested that this clustering results because many glycine substitutions may cause dominant lethal or sterile phenotypes.     

Evidence of in situ stability of the type IV collagen triple helix in human inflammatory bowel disease using a denaturation specific epitope antibody.

A peptide specific antibody (AH1OW1) was raised against an epitope, AH10 (aa 449-463), of the alpha1(IV) chain adjacent to a cleavage site for matrix metalloproteinases (MMP)-2 and -9 within the triple helix of type IV collagen. The antibody only reacted with denatured and reduced preparations of type IV collagen, or with pepsin isolated type IV collagen digested with MMP-2 and MMP-9. The specificity of this antibody for the denatured triple helix was demonstrated by the lack of staining with pre-immune antibody and by pre-incubation of AH1OW1 antibody with excess AH10 peptide epitope. The AH1OWI antibody was used to detect whether proteolysis of type IV collagen occurs in ulcerative colitis, an inflammatory bowel condition often characterised by a large influx of granulocytes and macrophages and an associated tissue destruction. However, no evidence of in situ proteolysis of the basement membrane type IV collagen was observed. Only in the most actively inflamed mucosa was staining with AH1OW1 antibody observed in the mucosal connective tissue. Digestion of frozen sections of bowel with MMP-1, MMP-2, MMP-3 and MMP-9 did not result in the exposure of the AH10 epitope. These data demonstrate the stability of intact type IV collagen and indicate that susceptibility of alpha1(IV) chain to digestion with MMP-2 and MMP-9 may require other proteolytic/denaturing events in the molecule.     

Decreased collagen stability as a response to the loss of structural integrity of thyroid cartilage

The thermal behavior, birefringence properties, and the biochemical composition of thyroid cartilage tissues have been studied. The hyaline cartilage, which was visualized as a quasi-isotropic medium, was composed of type II collagen, which did not denature at temperatures up to 100 degrees C. However, in hyaline cartilage digested by trypsin, the denaturation of collagen occured at 60 degrees C. Collagen fibers in the perichondrium were composed of type I and II collagen and formed a highly organized anisotropic structure (birefringence about 4.75 x 10(-3)) with a melting temperature of about 65 degrees C. The temperature of collagen denaturation in perichondrium in the whole system perichondrium-hyaline cartilage increased up to 75 degrees C, indicating the immobilization of perichondrium collagen by the extracellular matrix of the hyaline constituent.     

The incubation of laminin, collagen IV, and heparan sulfate proteoglycan at 35 degrees C yields basement membrane-like structures

Three basement membrane components, laminin, collagen IV, and heparan sulfate proteoglycan, were mixed and incubated at 35 degrees C for 1 h, during which a precipitate formed. Centrifugation yielded a pellet which was fixed in either potassium permanganate for ultrastructural studies, or in formaldehyde for Lowicryl embedding and immunolabeling with protein A-gold or anti-rabbit immunoglobulin-gold. Three types of structures were observed and called types A, B, and C. Type B consisted of 30-50-nm-wide strips that were dispersed or associated into a honeycomb-like pattern, but showed no similarity with basement membranes. Immunolabeling revealed that type B strips only contained heparan sulfate proteoglycan. The structure was attributed to self-assembly of this proteoglycan. Type A consisted of irregular strands of material that usually accumulated into semisolid groups. Like basement membrane, the strands contained laminin, collagen IV, and heparan sulfate proteoglycan, and, at high magnification, they appeared as a three-dimensional network of cord-like elements whose thickness averaged approximately 3 nm. But, unlike the neatly layered basement membranes, the type A strands were arranged in a random, disorderly manner. Type C structures were convoluted sheets composed of a uniform, dense, central layer which exhibited a few extensions on both surfaces and was similar in appearance and thickness to the lamina densa of basement membranes. Immunolabeling showed that laminin, collagen IV, and proteoglycan were colocalized in the type C sheets. At high magnification, the sheets appeared as a three-dimensional network of cords averaging approximately 3 nm. Hence, the organization, composition, and ultrastructure of type C sheets made them similar to the lamina densa of authentic basement membranes.     

Thermal stability of hepatitis B surface antigen S proteins.

Thermal stability of hepatitis B surface antigen (HBsAg) has been studied by analyzing alterations in the native secondary structure and the antigenic activity. After heating for 19 h, circular dichrosim showed a cooperative transition with a midpoint at 49 degrees C. The conformational changes induced by temperature reduced the helical content of HBsAg S proteins from 49% at 23 degrees C to 26% at 60 degrees C and abolished the antigenic activity, as measured by binding to polyclonal antibodies. Furthermore, the six different antigenic determinants recognized by our panel of monoclonal antibodies were also shown to be dependent on the native structure of HBsAg proteins. Hence, it can be inferred that these epitopes are conformation-dependent. Binding of monoclonal antibodies to HBsAg protected the native structure of the corresponding antigenic determinant from thermal denaturation. In fact, binding of one of the monoclonals tested resulted not only in protection of the corresponding epitope, but also in a consistent increase of antibody binding with increasing temperature. Such an increase in antibody binding occurred simultaneously with an increase in the fluidity of surface lipid regions, as monitored by fluorescence depolarization of 1-(trimethylammoniophenyl)-6-phenyl-1,3,5-hexatriene. This correlation, along with the observation that lipids play an important role in maintaining the structure and antigenic activity of HBsAg (Gavilanes et al. (1990) Biochem. J. 265, 857-864), allow to speculate the certain epitopes of HBsAg which are close to the lipid-protein interface, are dependent on the fluidity of the surface lipid regions. Thus, any change in the physical state of the lipids could confer a different degree of exposure to the antigenic determinants.     

Goodpasture disease. Characterization of a single conformational epitope as the target of pathogenic autoantibodies.

Goodpasture disease is a prototype autoimmune disease characterized by the formation of autoantibodies against the heterotrimeric basement membrane collagen type IV, which causes a rapidly progressive glomerulonephritis. The pathogenic antibody response is directed to the non-collagenous (NC1) domain of the alpha3 chain of type IV collagen (alpha3(IV)NC1), but not to the homologous region of the alpha1(IV)NC1. To identify the conformation-dependent immunodominant epitope on the alpha3(IV)NC1, a variety of recombinant NC1 domains were constructed by replacing single residues of alpha3(IV) with the corresponding amino acids from the nonreactive alpha1(IV) chain. Replacement mutations were identified that completely destroyed the Goodpasture epitope in the alpha3(IV) chain. Based on the identification of these critical positions, the epitope was finally reconstructed within the frame of the alpha1(IV) chain. The substitution of nine discontinuous positions in the alpha1(IV)NC1 with amino acid residues from the alpha3 chain resulted in a recombinant construct that was recognized by all patients' sera (n = 20) but by none of the sera from healthy controls (n = 10). This provides, for the first time, the molecular characterization of a single immunodominant conformational epitope recognized by pathogenic autoantibodies in a human autoimmune disease, representing the basis for the development of new epitope-specific strategies in the treatment of Goodpasture disease.     

Interactions of basement membrane components

The binding of laminin, type IV collagen, and heparan sulfate proteoglycan to each other was assessed. Laminin binds preferentially to native type IV (basement membrane) collagen over other collagens. A fragment of laminin (Mr 600 000) containing the three short chains (Mr 200 000) but lacking the long chain (Mr 400 000) showed the same affinity for type IV collagen as the intact protein. The heparan sulfate proteoglycan binds well to laminin and to type IV collagen. These studies show that laminin, type IV collagen and heparan sulfate proteoglycan interact with each other. Such interactions in situ may determine the structure of basement membranes.     

Characteristics of type IV collagen unfolding under various pH conditions as a model of pathological disorder in tissue

The overall structure of type IV collagen is the same at neutral and acidic pH, as determined by circular dichroism spectra. The heating rate dependence of denaturation midpoint temperature (T(m)) shows that type IV collagen is unstable at body temperature, similarly to type I collagen. The heating rate dependence of T(m) at neutral pH has two phases, but that at acidic pH apparently has a single phase. The T(m) of the first phase (lower T(m)) at neutral pH is consistent with that at acidic pH, and the activation energy of these phases is consistent, within experimental error. The triple helix region of type IV collagen corresponding to the second phase (higher T(m)) at neutral pH is thermally stable when compared to the triple helical structure at acidic pH. At acidic pH, as the loosely packed and unstable region has spread throughout the whole molecule, the thermal transition is thought to be cooperative and is observed as a single phase. Structural flexibility is related to protein function and assembly; therefore, the unstable structure and increased flexibility of type IV collagen induced at acidic pH may affect diseases accompanied by type IV collagen disorder.     

Characterization of a monoclonal antibody against human placenta type IV collagen by immunoelectroblotting, antibody-coupled affinity chromatography, and immunohistochemical localization

We have produced four monoclonal antibodies against type IV collagen obtained from human placenta. An antibody with a high titer by ELISA, named JK-199, reacted not only with type IV collagen in the triple-helical conformation but also with thermally denatured chains. After affinity chromatography on JK-199 antibody-coupled resin, the amino acid composition and CD spectrum of the affinity-purified peptides from the crude pepsin extract of human placenta were typical of those of human type IV collagen in the triple-helical conformation. On SDS-polyacrylamide gel electrophoresis, the purified protein showed only one broad band with a molecular weight of approximately 260,000 before reduction and six smaller peptide bands after reduction. On immunoelectroblotting, JK-199 reacted with all six peptide bands. Immunohistochemically, typical basement membranes were exclusively and strongly stained with JK-199 on frozen sections of PLP-fixed human placentas without any enzymatic pretreatment in the routine immunoperoxidase method. Judging from these findings, it is concluded that the epitopes of type IV collagen that reacted with JK-199 are exposed on the surface of basement membranes. This antibody should be useful for identification of type IV collagen in normal or pathological basement membranes or other structures.     

Interactions of basement membrane components

The binding of laminin, type IV collagen, and heparan sulfate proteoglycan to each other was assessed. Laminin binds preferentially to native type IV (basement membrane) collagen over other collagens. A fragment of laminin (M_r 600 000) containing the three short chains (M_r 200 000) but lacking the long chain M_r 400 000) showed the same affinity for type IV collagen as the intact protein. The heparan sulfate proteoglycan binds well to laminin and to type IV collagen. These studies show that laminin, type IV collagen and heparan sulfate proteoglycan interact with each other. Such interactions in situ may determine the structure of basement membranes.     

Effects of thermal exposure on immunophenotyping combined with in situ PCR, measured by flow cytometry.

BACKGROUND: The combination of in situ PCR and cell phenotyping by antibody labeling (ISPCR/Flow) allows for the identification of cell subsets carrying a particular genetic sequence. ISPCR utilizes thermal cycling for genetic amplification, which can reduce the effectiveness of surface antibody labeling. This study explored and characterized the effects of thermal exposure on antibody labeling using CD4 and CD45. METHODS: Single temperature incubations and thermal cycling exposures were performed on leukocytes labeled with either direct antibody conjugates or with biotinylated antibodies and PE-streptavidin. RESULTS: Fluorescence emission decreased above 70 degrees ( )C when cells were stained with directly conjugated antibodies or a biotinylated antibody and PE-streptavidin prior to high heat exposure. If counter stained with PE-streptavidin after heat, fluorochrome fluorescence was detectable. We tested a second CD4 clone, that provided poor results under similar labeling conditions, suggesting the combination of fixation and heat may have an epitope specific effect for the same cellular antigen. CONCLUSIONS: Immunophenotyping can be combined with ISPCR, but each antibody must be tested to determine its efficacy. The denaturation of protein above 70 degrees C appears to be the main reason for loss of fluorescence. The best procedure is to first stain cells with a biotinylated antibody to an epitope that survives fixation and thermocycling. The cells are then subjected to the desired PCR procedure. Finally they are stained with a fluorochrome conjugated streptavidin.     

Physical and immunochemical studies of the globular domain of type IV collagen. Cryptic properties of the Goodpasture antigen

The globular domain of type IV collagen from bovine glomerular basement membrane was isolated under nondenaturing conditions. It was shown to exist in a hexameric form comprising monomeric and dimeric subunits, with the Goodpasture antigen residing in monomer M2 and dimer D2 as previously described (Butkowski, R. J., Wieslander, J., Wisdom, B. J., Barr, J. F., Noelken, M. E., and Hudson, B. G. (1985) J. Biol. Chem. 260, 3739-3747). The epitope, however, is sequestered inside the hexamer, but becomes exposed and binds with the Goodpasture antibody upon dissociation of the hexamer into its subunits after treatment with concentrated guanidine HC1 or dilute acetic acid (pH less than 3.0). The process is completely reversible even from the denatured state. Circular dichroism studies show that the conformation of each subunit is unusually resistant to change in 6 M guanidine HC1 at 25 degrees C. This suggests that exposure of the epitope by dissociation requires minimal or no unfolding of subunits. The results provide additional evidence for localization of the Goodpasture antigen to the globular domain of type IV collagen. Moreover, these studies extend the conclusion (Weber, H., Engel, J., Wiedemann, H., Glanville, R., and Timpl, R. (1984) Eur. J. Biochem. 139, 401-410) about a tumor basement membrane, to an authentic physiological membrane, that the globular domain is a major cross-linking site in the type IV collagen matrix.     

Assembly of type IV collagen. Insights from alpha3(IV) collagen-deficient mice

Type IV collagen includes six genetically distinct polypeptides named alpha1(IV) through alpha6(IV). These isoforms are speculated to organize themselves into unique networks providing mammalian basement membranes specificity and inequality. Recent studies using bovine and human glomerular and testis basement membranes have shown that unique networks of collagen comprising either alpha1 and alpha2 chains or alpha3, alpha4, and alpha5 chains can be identified. These studies have suggested that assembly of alpha5 chain into type IV collagen network is dependent on alpha3 expression where both chains are normally present in the tissue. In the present study, we show that in the lens and inner ear of normal mice, expression of alpha1, alpha2, alpha3, alpha4, and alpha5 chains of type IV collagen can be detected using alpha chain-specific antibodies. In the alpha3(IV) collagen-deficient mice, only the expression of alpha1, alpha2, and alpha5 chains of type IV collagen was detectable. The non-collagenous 1 domain of alpha5 chain was associated with alpha1 in the non-collagenous 1 domain hexamer structure, suggesting that network incorporation of alpha5 is possible in the absence of the alpha3 chain in these tissues. The present study proves that expression of alpha5 is not dependent on the expression of alpha3 chain in these tissues and that alpha5 chain can assemble into basement membranes in the absence of alpha3 chain. These findings support the notion that type IV collagen assembly may be regulated by tissue-specific factors.     

Proteolytic exposure of a cryptic site within collagen type IV is required for angiogenesis and tumor growth in vivo

Evidence is provided that proteolytic cleavage of collagen type IV results in the exposure of a functionally important cryptic site hidden within its triple helical structure. Exposure of this cryptic site was associated with angiogenic, but not quiescent, blood vessels and was required for angiogenesis in vivo. Exposure of the HUIV26 epitope was associated with a loss of alpha1beta1 integrin binding and the gain of alphavbeta3 binding. A monoclonal antibody (HUIV26) directed to this site disrupts integrin-dependent endothelial cell interactions and potently inhibits angiogenesis and tumor growth. Together, these studies suggest a novel mechanism by which proteolysis contributes to angiogenesis by exposing hidden regulatory elements within matrix-immobilized collagen type IV.     

Immunohistochemical localization of type IV collagen fibronectin and laminin in the juxtaglomerular apparatus of the rat kidney

The juxtaglomerular apparatus (JGA) is a complex structure containing several components: the vessels, the extraglomerular mesangium and the distal tubule. These structures include cellular elements and an extracellular matrix (ECM). Collagenous (type IV collagen) and noncollagenous components of the basement membranes were studied. The localization of type IV collagen and of two extracellular glycoproteins (laminin and fibronectin) was investigated using immunofluorescent and immunoperoxidase labelled antibodies. Type IV collagen and laminin have the same localization on the JGA basement membranes. On the other hand, fibronectin is limited to the entrance of the glomerular stalk. On electron microscopy, type IV collagen is found in the basement membrane while fibronectin is restricted to certain areas of the extracellular matrix. These findings confirm data concerning the distribution of these three components in basement membranes and allow a better understanding of the histoarchitecture of the juxtaglomerular apparatus.     

Conformational features of a natural break in the type IV collagen Gly-X-Y repeat

Fibrillar collagens have an absolute requirement for Gly as every 3rd residue, whereas breaks in the Gly-X-Y repeating pattern are found normally in the triple helix domains of non-fibrillar collagens, such as type IV collagen in basement membranes. In this study, a model 30-mer peptide is designed to include the interruption GPOGAAVMGPOGPO found in the alpha5 chain of type IV collagen. The GAAVM peptide forms a stable triple helix, with Tm= 29 degrees C. When compared with a control peptide with Gly as every 3rd residue, the GAAVM peptide has a marked decrease in the 225 nm maximum of its CD spectrum and a 10 degrees C drop in stability. A 50% decrease in calorimetric enthalpy is observed, which may result from disruption of ordered water structure anchored by regularly placed backbone carbonyls. NMR studies on specific 15N-labeled residues within the GAAVM peptide indicate a normal triple helical structure for Gly-Pro-Hyp residues flanking the break. The sequence within the break is not disordered but shows altered hydrogen exchange rates and an abnormal Val chemical shift. It was previously reported that a peptide designed to model a similar kind of interruption in the peptide (Pro-Hyp-Gly)10, (GPOGPOPOGPO), is unable to form a stable triple helix, and replacement of GAA by GPO or VM by PO within the GAAVM break decreases the stability. Thus, rigid imino acids are unfavorable within a break, despite their favorable stabilization of the triple helix itself. These results suggest some non-random structure typical of this category of breaks in the Gly-X-Y repeat of the triple helix.     

Type IV Collagen Is Detectable in Most, but Not All, Basement Membranes of Caenorhabditis elegans and Assembles on Tissues That Do Not Express It

Type IV collagen in Caenorhabditis elegans is produced by two essential genes, emb-9 and let-2, which encode α1- and α2-like chains, respectively. The distribution of EMB-9 and LET-2 chains has been characterized using chain-specific antisera. The chains colocalize, suggesting that they may function in a single heterotrimeric collagen molecule. Type IV collagen is detected in all basement membranes except those on the pseudocoelomic face of body wall muscle and on the regions of the hypodermis between body wall muscle quadrants, indicating that there are major structural differences between some basement membranes in C. elegans. Using lacZ/green fluorescent protein (GFP) reporter constructs, both type IV collagen genes were shown to be expressed in the same cells, primarily body wall muscles, and some somatic cells of the gonad. Although the pharynx and intestine are covered with basement membranes that contain type IV collagen, these tissues do not express either type IV collagen gene. Using an epitope-tagged emb-9 construct, we show that type IV collagen made in body wall muscle cells can assemble into the pharyngeal, intestinal, and gonadal basement membranes. Additionally, we show that expression of functional type IV collagen only in body wall muscle cells is sufficient for C. elegans to complete development and be partially fertile. Since type IV collagen secreted from muscle cells only assembles into some of the basement membranes that it has access to, there must be a mechanism regulating its assembly. We propose that interaction with a cell surface–associated molecule(s) is required to facilitate type IV collagen assembly.     

Thermal analysis of CHL V79 cells using differential scanning calorimetry: implications for hyperthermic cell killing and the heat shock response

Differential scanning calorimetry (DSC) was used to assay thermal transitions that might be responsible for cell death and other responses to hyperthermia or heat shock, such as induction of heat shock proteins (HSP), in whole Chinese hamster lung V79 cells. Seven distinct peaks, six of which are irreversible, with transition temperatures from 49.5 degrees C to 98.9 degrees C are detectable. These primarily represent protein denaturation with minor contributions from DNA and RNA melting. The onset temperature of denaturation, 38.7 degrees C, is shifted to higher temperatures by prior heat shock at 43 degrees and 45 degrees C, indicative of irreversible denaturation occurring at these temperatures. Thus, using DSC it is possible to demonstrate significant denaturation in a mammalian cell line at temperatures and times of exposure sufficient to induce hyperthermic damage and HSP synthesis. A model was developed based on the assumption that the rate limiting step of hyperthermic cell killing is the denaturation of a critical target. A transition temperature of 46.3 degrees C is predicted for the critical target in V79 cells. No distinct transition is detectable by DSC at this temperature, implying that the critical target comprises a small fraction of total denaturable material. The short chain alcohols methanol, ethanol, isopropanol, and t-butanol are known hyperthermic sensitizers and ethanol is an inducer of HSP synthesis. These compounds non-specifically lower the denaturation temperature of cellular protein. Glycerol, a hyperthermic protector, non-specifically raises the denaturation temperature for proteins denaturing below 60 degrees C. Thus, there is a correlation between the effect of these compounds on protein denaturation in vivo and their effect on cellular sensitivity to hyperthermia.