Mutational analysis of type IV collagen alpha5 chain, with respect to heterotrimer formation

Alport syndrome (AS) is caused by mutations in type IV collagen α3, α4, and α5 chains. The three chains form a heterotrimer. In this study, we introduced 12 kinds of missense and three kinds of nonsense mutations, corresponding to AS mutations, into the NC1 domain of α5(IV) and characterized the mutant chains. Nine α5(IV) chains with amino acid substitutions and all three truncated α5(IV) chains did not form a heterotrimer and were not secreted from cells. Three α5(IV) chains with amino acid substitutions did, however, form heterotrimers in cells, but these were not secreted from cells. These findings indicate that a defect in heterotrimer formation is the main molecular mechanism underlying the pathogenesis of AS caused by mutation in the NC1 domain. We also showed that even a single amino acid deletion in the carboxyl-terminal region markedly affected the heterotrimerization, indicating that the carboxyl-terminal end is indispensable for heterotrimer formation.     

Specific binding of collagen type IV to Streptococcus pyogenes

Many strains of Streptococcus pyogenes are capable of binding type IV collagen. In the present study, all 50 S. pyogenes strains isolated from patients with acute glomerulonephritis showed high or moderate affinity for radiolabelled type IV collagen. A majority of strains of other sources, such as reference strains of various M-types and strains isolated from patients with pharyngeal infections also bound type IV collagen; however, a number of weak binders or non-binders were found among those. The collagen type IV binding component(s) on S. pyogenes were susceptible to proteinase K digestion, partially sensitive to trypsin but insensitive to pepsin treatment at pH 5.5. According to tests with three M-positive strains and their M-negative derivatives, the binding was not dependent on M-protein. The binding was saturable with time and inhibited by unlabelled type IV collagen. Partially inhibition was found with type II collagen, gelatin and fibrinogen but not with a number of other serum proteins.     

Differential expression of type IV collagen isoforms in rat glomerular endothelial and mesangial cells

Type IV collagen, which is encoded by six genetically distinct alpha-chains (alpha 1-alpha 6), is a major component of the kidney glomerulus. The alpha 1(IV) and alpha 2(IV) chains are present predominantly in the mesangial matrix, whereas the alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains are localized almost exclusively to the glomerular basement membrane (GBM). Thickening of the GBM and expansion of the mesangial matrix are believed to contribute to the pathogenesis of diabetic nephropathy. In the present study, we evaluated the expression of alpha 1(IV), alpha 3(IV), and alpha 5(IV) chains in rat glomerular endothelial (GEndC) and mesangial cells (GMC). Under physiological concentrations of glucose (5 mM), alpha 1(IV) and alpha 5(IV) chains were detectable in GMCs, with an obvious absence of alpha 3(IV) chain. All three isoforms tested were present in GEndCs. At diabetic concentrations of glucose (25 mM), alpha 1(IV) was up-regulated in GMCs, whereas expression level of alpha 1(IV) remained unaltered in GEndCs. The alpha 3(IV) and alpha 5(IV) chains were up-regulated in GEndCs, but remained unchanged in GMCs under diabetic glucose concentrations (25 mM). Collectively, our results demonstrate that GMC might contribute to mesangial matrix expansion, mediated by alpha 1(IV) collagen, while GEndC might contribute to thickening of GBM, mediated by alpha 3(IV) collagen, in patients with diabetic nephropathy.     

The function of type IV collagen during Drosophila embryogenesis

A collagen gene (Dcg1) was characterized in Drosophila melanogaster and shown to encode a peptide related to vertebrate basement membrane type IV collagen chains. To study the function of type IV collagen during Drosophila development, we transformed flies with a partially truncated Dcg1 gene under the control of a heat-shock promotor. This construct induced synthesis of shortened pro- chains which associated with normal ones and thereby caused degradation of the shortened and normal pro- chains through a process called pro-collagen suicide. A large proportion of embryos expressing the transgene developed a phenotype exhibiting absence or partial retraction of the germ band with defects in nerve cord condensation and dorsal closure. Together these results indicated that, during embryogenesis, type IV collagen was an essential guiding factor for cell-matrix interactions in morphogenetic events.     

Distribution of laminin and types IV and III collagen in fetal,infant and adult human spleens

Summary The immunohistochemical distribution of the basement membrane (BM) proteins, laminin and type IV collagen, and interstitial type III collagen was investigated in 12 fetal spleens at the 15th–38th gestational weeks (g.w.) and in spleens of 8 infants from term to 4 years. The results were compared with the distribution of the same proteins in adult human spleen. BM proteins were found to be abundantly present in the red pulp of all spleens during the whole of development. The content of type III collagen gradually decreased with advancing age and, in adult spleen, there were only occasional positively staining fibers in Billroth's cords. This finding indicates that the composition of reticular fibers in the red pulp of spleen is different from the reticular fibers elsewhere in lymphoreticular tissue. Early signs of ring fiber formation in the walls of venous sinuses were detectable at the 15th–19th g.w., although their more complete development occurred relatively late from the 36th g.w. onwards. Ring fibers contained both laminin and type IV collagen in all the investigated spleens. They never stained for type III collagen. The developing white pulp was positive for BM proteins, but showed no staining for type III collagen at the 15th g.w. At later ages, the white pulp stained similarly for both BM proteins and type III collagen.     

Structural organization of the gene for the alpha 1 chain of human type IV collagen

The complete exon size and distribution pattern in the gene for the alpha 1 chain of human type IV collagen was determined. Clones covering 145 kilobases (kb) of genomic DNA including 100 kb of the gene itself as well as 25 kb upstream and 20 kb downstream of the gene sequences, respectively, were isolated from lambda phage and cosmid libraries. The overall gene structure was determined by endonuclease restriction mapping and R-loop analyses and all exon sizes by nucleotide sequencing. The characterized clones contained all the coding sequences except for exon 2 whose sequence was determined after its amplification by the polymerase chain reaction. There were four gaps in the intron sequences; the exact size of the gene is unknown. The entire gene is at least 100 kb in size and contains 52 exons whose size distribution is completely different from that of the genes for fibrillar collagens. In the -Gly-X-Y- coding region there are three exons of 99, 90, and 45 base pairs (bp) each and two exons of 27, 36, 42, 51, 54, 63, and 84 bp each. The rest of the exons have sizes between 71 and 192 bp in the collagenous region. About one-half of the -Gly-X-Y- repeat coding exons start with the second base for the codon of glycine, whereas the other half starts (with two exceptions) with a complete glycine codon. The distribution of split versus unsplit codons is uneven in that the first 19 exons of the gene start with a complete codon. The gene contains repetitive sequences in several regions. A 185-nucleotide segment containing 40 copies of CCT flanked by poly(C) and poly(T) sequences was shown to be located adjacent to an exon. The gene has previously been shown to be located head-to-head to the alpha 2(IV) collagen gene at the distal end of the long arm of chromosome 13, such that the first exons of the two genes are separated by as little as 42 bp (P?schl, E., Pollner, R., and Kühn, K. (1988) EMBOJ. 7,2687-2695; Soininen, R., Huotari, M., Hostikka, S. L., Prockop, D. J., and Tryggvason, K. (1988) J. Biol. Chem. 263, 17217-17220). The results demonstrate that the human alpha 1(IV) collagen gene has a structure distinctly different from the genes for fibrillar collagens and also that it is considerably larger than any collagen gene characterized to date.     

Molecular mobility in the gap regions of type 1 collagen fibrils

Recent studies of the structure of Type I collagen fibrils (Piez and Trus,Biosci. Rep.1:801–810, 1981; Fraser, MacRae, Miller and Suzuki,J. Mol. Biol.167:497–521, 1983) suggest that the segments of the collagen molecule which comprise the gap region are more mobile than those which comprise the overlap region. We have analyzed the distribution of amino acid residues and triplet types between the two regions, and find significantly non-uniform distributions for Ala, Gln, His, Hyp, Leu, Phe, and Tyr, and for triplets containing two imino acid residues. Taken together with the lower packing density in the gap region these observations provide a basis for understanding the greater mobility of the molecular segments in the gap region. In addition, we have examined the linear distribution of residue types in the two regions and also the hydropathy profile (Kyte and Doolittle,J. Mol. Biol.157: 105–113, 1982). These reveal a segment of the gap region comprising helical residues 165–173, 399–407, 633–641 and 867–975 which has the highest hydropathy index, is devoid of charged residues, and contains very high proportions of Ala, Hyp and Phe.     

The complete primary structure of the alpha 2 chain of human type IV collagen and comparison with the alpha 1(IV) chain

The complete primary structure of the human type IV collagen alpha 2(IV) chain has been determined by nucleotide sequencing of cDNA clones. The overlapping cDNA clones cover 6,257 base pairs with a 5'-untranslated region of 283 base pairs, the 5,136-base pair open reading frame, and the 3'-untranslated region of 838 base pairs. The predicted amino acid sequence demonstrates that the complete translation product consists of 1,712 residues corresponding in molecular weight to 167,560. The translated polypeptide has a signal peptide of 36 amino acids, an amino-terminal noncollagenous part of 21 residues, a 1,428-residue collagenous domain with 23 interruptions, and a carboxyl-terminal noncollagenous (NC) domain of 227 residues. The calculated molecular mass of the mature human alpha 2(IV) chain is 163,774 Da.     

Sequence and localization of a partial cDNA encoding the human alpha 3 chain of type IV collagen.

A novel type IV collagen, alpha 3(IV), has recently been identified in human and bovine basement membranes. Here we describe the cloning and sequencing of a cDNA encoding 218 residues of the NC1 domain of the human alpha 3(IV) chain. Of interest is the possible role of abnormalities of the alpha 3(IV) chain in Alport syndrome, as suggested by the failure to detect the NC1 domain of alpha 3(IV) in the basement membranes of some Alport syndrome patients. To determine whether the alpha 3(IV) gene (COL4A3) may be mutated in Alport syndrome, we localized it, by somatic cell hybrid analysis and in situ hybridization of metaphase chromosomes, to chromosome 2q35-2q37. Mutations in alpha 3(IV) cannot therefore be responsible for the vast majority of cases of Alport syndrome, which have been shown to be X linked. One explanation for the immunochemical data implicating alpha 3(IV) in Alport syndrome pathogenesis is that mutations of the alpha 5(IV) chain, which has been localized to Xq22 and found to be mutated in at least three kindreds with Alport syndrome, lead to failure to incorporate the alpha 3(IV) chains into the multimeric structure of glomerular basement membrane in a stable fashion.     

Analysis of the role of the COL1 domain and its adjacent cysteine-containing sequence in the chain assembly of type IX collagen

The mechanisms of chain selection and assembly of type IX collagen, a heterotrimer 1(IX)2(IX)3(IX), must differ from that of fibrillar collagens since it lacks the characteristic C-propeptide of these latter molecules. We have tested the hypothesis that the information required for this process is contained within the C-terminal triple helical disulfide-bonded region (LMW). The reassociations of the purified LMW fragments of pepsinized bovine type IX collagen were followed by the formation of disulfide-bonded multimers. Our data demonstrate that only three triple helical assemblies form readily, (1)₃, (2)₃, and 123. The information required for chain selection and assembly is thus, at least in part, contained in the studied fragments. Molecular stoichiometries different from the classical heterotrimer may thus also form under certain conditions.     

Cloning, purification, and characterization of a non-collagenous anti-angiogenic protein domain from human alpha1 type IV collagen expressed in Sf9 cells

alpha1(IV)NC1, a cleavage fragment of the carboxy terminal non-collagenous human alpha1 chain of type IV collagen, is derived from the extracellular matrix specifically by MMP-2. Recently we determined the in vitro and in vivo anti-angiogenic activity of alpha1(IV)NC1 and presently, its role in cancer therapy is under evaluation. To characterize alpha1(IV)NC1 as a potential candidate for drug development and to test its efficacy in animal models, an effective method to produce a purified active form of alpha1(IV)NC1 is needed. In the present study, expression of alpha1(IV)NC1 in Sf9 cells using baculovirus expression system was discussed, this method was found to be effective in the production of a functionally active soluble form of the recombinant protein. The purified protein showed its characteristic activities such as inhibiting cell proliferation, migration, and tube formation in endothelial cells.     

Preparation and characterization of polyclonal and monoclonal antibodies against human interleukin 1 alpha (IL 1 alpha)

Polyclonal and monoclonal anti-human IL 1 alpha antibodies (Ab) have been established. These Ab neutralized human recombinant IL 1 alpha (rIL 1 alpha) activity effectively, but did not interfere with human rIL 1 beta, murine rIL 1 alpha, or human rIL 2 activity. Fifty percent of rIL 1 alpha activity (25 U/ml, or 2.5 ng/ml) was neutralized by less than 0.06 microgram/ml of rabbit anti-IL 1 alpha Ab (R-38.3G) and by less than 0.13 microgram/ml of monoclonal Ab (clone 28(3B1], respectively. In other experiments, 10 micrograms/ml of rabbit anti-IL 1 alpha Ab could effectively neutralize 50% of 2000 U of rIL 1 alpha activity, and the same amount of monoclonal Ab neutralized 50% of 500 U/ml of rIL 1 alpha activity. Not only IL 1 alpha activity in the thymocyte costimulator assay, but also IL 1-dependent IL 2 production by a human leukemic cell line, HSB.2 subclone, were blocked by these polyclonal or monoclonal Ab. In addition, pI 4.9 IL 1 activity produced by the myelomonocytic cell line THP-1 and by the Epstein-Barr virus-transformed B cell lines, were neutralized by these Ab, suggesting that these cell lines also produce IL 1 alpha. The specificity of these polyclonal and monoclonal Ab was further confirmed by immunochemical method (Western blotting), in which anti-IL 1 alpha Ab reacted with rIL 1 alpha in a specific manner. Furthermore, an enzyme-linked immunosorbent assay system has been developed that can detect low levels of IL 1 alpha activity (less than 0.3 ng/ml or less than 3 U/ml), which is still less sensitive than thymocyte comitogenic assay and considerably less sensitive than the D10 assay. Finally, anti-IL 1 alpha Ab-conjugated affinity columns were prepared, by which IL 1 alpha activity, but not IL 1 beta activity, was specifically adsorbed and eluted effectively.     

Production and partial characterization of monoclonal antibodies specific for the gamonts of Eimeria tenella.

Thirteen hybridomas secreting monoclonal antibodies (Mabs) specific for the sexual stages (gamonts) of Eimeria tenella were produced by fusing spleen cells of gamont-immunized RBF/Dn mice with FOX-NY myeloma cells. A Mab subisotype profile revealed 1 IgG2a and 12 IgG1. All Mabs demonstrated a similar binding pattern when incubated with parasitic gamonts as determined by the indirect fluorescent antibody test. Ascitic fluid containing Mab (GD9 (IgG1) was produced and used to immunize chicks passively per os. There was a 34% decrease (P less than 0.05) in oocyst output from immunized chicks when compared to control chicks. Passively immunized chicks also had reduced cecal lesion scores when compared to control chicks. These results suggest that Mab GD9 partially inhibited the fertilization process of E. tenella.     

Human basement membrane collagen (type IV). The amino acid sequence of the alpha 2(IV) chain and its comparison with the alpha 1(IV) chain reveals deletions in the alpha 1(IV) chain

The cDNA and protein sequences of the N-terminal 60% of the alpha 2(IV) chain of human basement membrane collagen have been determined. By repeated primer extension with synthetic oligodeoxynucleotides and mRNA from either HT1080 cells or human placenta overlapping clones were obtained which cover 3414 bp. The derived protein sequence allows for the first time a comparison and alignment of both alpha chains of type IV collagen from the N terminus. This alignment reveals an additional 43 amino acid residues in the alpha 2(IV) chain as compared to the alpha 1(IV) chain. 21 of these additional residues form a disulfide-bridged loop within the triple helix which is unique among all known collagens.     

Preparation and characterization of monoclonal antibodies specific for GoCodGdCGoCo duplex with high-anti conformation

Monoclonal antibodies were prepared using a self-complementary oligonucleotide duplex containing cyclonucleosides with the high-anti conformation as an antigen. A competition ELISA assay showed that the monoclonal antibodies specifically recognized high-anti left-handed oligonucleotides but not oligonucleotides with B- or Z-conformation.     

Purification and characterization of a transgenic corn grain‐derived recombinant collagen type I alpha 1

Corn offers advantages as a transgenic host for producing recombinant proteins required at large volumes (1,000's of tons per year) and low cost (less than US$50/kg) by generating them as co‐products of biorefining. We describe the purification and characterization of a corn grain‐derived mammalian structural protein having such market characteristics: a full length recombinant collagen type I alpha 1 (rCIα1) chain. Material properties of interest are gelation behavior, which would depend on as yet unverified ability of corn to carry out post‐translational prolyl hydroxylation and formation of triple helical conformation. The starting material was grain where the expression of rCIα1 had been directed by an embryo‐specific promoter. Purification consisted of extraction at low pH followed by membrane and chromatographic steps to isolate rCIα1 for characterization. The amino acid composition and immunoreactivity of CIα1 was similar to that of an analogous native human CIα1 and to rCIα1 produced by the yeast Pichia pastoris. Tandem mass spectrometry confirmed the primary sequence of the corn‐derived rCIα1 with 46% coverage. Fragments of the rCIα1chains were also observed, possibly caused by endogenous plant proteases. The corn‐derived rCIα1 had a low level of prolyl hydroxylation (～1% versus 11%) relative to animal‐derived CIα1 and folded into its characteristic triple‐helical structure as indicated by its resistance to pepsin digestion below its melting temperature of 26^oC. The 29 amino acid foldon fused to the C‐terminus to initiate triple helix formation was not cleaved from the rCIα1chains, but could be removed by pepsin treatment. © 2009 American Institute of Chemical Engineers Biotechnol. Prog. 2009     

The structure of avian type XII collagen. Alpha 1 (XII) chains contain 190-kDa non-triple helical amino-terminal domains and form homotrimeric molecules

The monoclonal antibody 75d7, specific for type XII collagen (Sugrue, S.P., Gordon, M.K., Seyer, J., Dublet, B., van der Rest, M., and Olsen, B. R. (1989) J. Cell Biol., in press), was used to characterize the intact form of type XII collagen from chick embryo leg tendons. On an immunoblot of a 6% polyacrylamide gel of tendon extracts, one sharp band is recognized by the antibody at Mr = 220,000, while two fuzzy and poorly resolved bands are seen at Mr = 270,000 and Mr = 290,000. By immunoprecipitation of radiolabeled tendon culture media and electrophoresis of the precipitated material, bands with the same mobilities are observed, indicating that type XII collagen is not proteolytically processed in the extracellular space. Type XII collagen was extracted from tendons with 1 M NaCl in a Tris-HCl buffer and partially purified by concanavalin A-Sepharose and gel permeation chromatographies, using dot immunoblots to monitor the purification. Fractions highly enriched in bacterial collagenase-sensitive proteins with the same electrophoretic properties as type XII collagen were obtained. These fractions did not stain with Alcian blue and neither they nor the immunostained type XII collagen were affected by chondroitinase ABC digestion, indicating that type XII collagen is not a proteoglycan. A disulfide-bonded trimeric CNBr peptide was isolated by affinity chromatography on an antibody column and further purified by gel electrophoresis. Its NH2-terminal amino acid sequence was shown to be unique, demonstrating that type XII collagen is a homotrimer [alpha 1 (XII)]3. After bacterial collagenase digestion, both the immunopurified radiolabeled preparation and the purified tendon extract fraction showed by gel electrophoresis the presence of a large disulfide-bonded, 3 x 190-kDa, collagenase-resistant domain. Rotary shadowing and electron microscopy of the purified type XII fraction demonstrated that the molecule has the structure of a cross consisting of a 75 nm collagenase-sensitive tail, a central globule, and three 60 nm arms each ending in a small globule. After heat denaturation and renaturation, only a very large globule can be seen, attached to the triple helical tail. These results show that type XII collagen has a unique structure and is different from the other matrix constituents described so far.     

Isolation and characterization of human monoclonal antibodies specific to MMP-1A, MMP-2 and MMP-3

Matrix metalloproteinases (MMPs), a group of more than 20 zinc-containing endopeptidases, are up-regulated in many diseases, but the use of MMP inhibitors for therapeutic purposes has often been disappointing, possibly for the limited specificity of the drugs used in clinical trials. In principle, individual MMPs could be specifically drugged by monoclonal antibodies, either by inhibition of their catalytic activity or by antibody-based pharmacodelivery strategies. In this article we describe the isolation and affinity maturation of recombinant antibodies (SP1, SP2, SP3) specific to the murine catalytic domains of MMP-1A, MMP-2 and MMP-3. These novel reagents allowed a systematic comparative immunofluorescence analysis of the expression patterns of their cognate antigens in a variety of healthy, cancerous and arthritic murine tissues. While all three MMPs were strongly expressed in tumor and arthritis specimens, MMP-1A was completely undetectable in the normal tissues tested, while MMP-2 and MMP-3 exhibited a weak expression in certain normal tissues (e.g., liver). The new antibodies may serve as building blocks for the development of antibody-based therapy strategies in mouse models of pathology.     

Expression of type IV collagen-degrading activity during early embryonal development in the sea urchin and the arresting effects of collagen synthesis inhibitors on embryogenesis

Type IV collagen-degrading activity was expressed in homogenates of Lytechinus pictus embryos during embryogenesis. Activity was concentrated 1,600-fold by ammonium sulfate fractionation, ion exchange, and gel chromatography and could not be activated further upon trypsin or organomercurial treatment. This enzyme activity could also degrade gelatin but had no affinity for type I, III, and V collagens. Activity was inhibited by addition of excess type IV collagen or gelatin, but was unaffected by addition of excess amounts of non-collagenous proteins of the extracellular matrix. Chelators such as 1,10-phenanthroline or Na₂EDTA reduced activity to control levels. Inhibitors of plasmin and of serine and thiol proteases were without effect. Type IV collagen-degrading activity first became apparent at the stage of early mesenchyme blastula. It then increased by a small increment and remained stable up to the stage of late mesenchyme blastula, coinciding with first detection of collagen synthesis and the appearance of the archenteron. Thereafter, a sharp increase in activity was observed, concurrently with remodelling of the archenteron. Maximum activity was attained at prism stage and was retained throughout to pluteus-larva stage. The specific inhibitors of collagen biosynthesis 8,9-dihydroxy-7-methyl-benzo[b]quinolizinium bromide and tricyclodecane-9-yl xanthate arrested sea urchin embryo development at early blastula, prevented the invagination of the archenteron, and reverted the expression of type IV collagen-degrading activity to non-detectable levels. Removal of the inhibitors allowed embryos to gastrulate and express type IV collagen-degrading activity.