Triple-helical Molecules Research Articles

Genetic reprogramming with stem cells regenerates glomerular epithelial podocytes in Alport syndrome.

Glomerular filtration relies on the type IV collagen (ColIV) network of the glomerular basement membrane, namely, in the triple helical molecules containing the α3, α4, and α5 chains of ColIV. Loss of function mutations in the genes encoding these chains (Col4a3, Col4a4, and Col4a5) is associated with the loss of renal function observed in Alport syndrome (AS). Precise understanding of the cellular basis for the patho-mechanism remains unknown and a specific therapy for this disease does not currently exist. Here, we generated a novel allele for the conditional deletion of Col4a3 in different glomerular cell types in mice. We found that podocytes specifically generate α3 chains in the developing glomerular basement membrane, and that its absence is sufficient to impair glomerular filtration as seen in AS. Next, we show that horizontal gene transfer, enhanced by TGFβ1 and using allogenic bone marrow-derived mesenchymal stem cells and induced pluripotent stem cells, rescues Col4a3 expression and revive kidney function in Col4a3-deficient AS mice. Our proof-of-concept study supports that horizontal gene transfer such as cell fusion enables cell-based therapy in Alport syndrome.

Instrument for tensile testing of individual collagen fibrils with facile sample coupling and uncoupling.

Collagen is the major structural protein in human bodies constituting about 30% of the entire protein mass. Through a self-assembly process, triple helical collagen molecules assemble into high aspect-ratio fibers of tens to hundreds of nanometer diameter, known as collagen fibrils (CFs). In the last decade, several methods for tensile testing these CFs emerged. However, these methods are either overly time-consuming or offer low data acquisition bandwidth, rendering dynamic investigation of tensile properties impossible. Here, we describe a novel instrument for tensile testing of individual CFs. CFs are furnished with magnetic beads using a custom magnetic tweezer. Subsequently, CFs are lifted by magnetic force, allowing them to be picked-up by a microgripper structure, which is mounted on a cantilever-based interferometric force probe. A piezo-lever actuator is used to apply tensile displacements and to perform tensile tests of tethered CFs, after alignment. Once the mechanical tests are finished, CFs are removed from the microgripper by application of a magnetic field. Our novel instrument enables tensile tests with at least 25-fold increased throughput compared to tensile testing with an atomic force microscope while achieving force resolution (p-p) of 10 nN at a strain resolution better than 0.1%.

Preservation of collagen in the soft tissues of frozen mammoths

We investigated the characteristics of extracellular matrix (ECM) in the soft tissue of two frozen baby woolly mammoths (Mammuthus primigenius) that died and were buried in Siberian permafrost approximately 40,000 years ago. Morphological and biochemical analyses of mammoth lung and liver demonstrated that those soft tissues were preserved at the gross anatomical and histological levels. The ultrastructure of ECM components, namely a fibrillar structure with a collagen-characteristic pattern of cross-striation, was clearly visible with transmission and scanning electron microscopy. Type I and type IV collagens were detected by immunohistochemical observation. Quantitative amino acid analysis of liver and lung tissues of the baby mammoths indicated that collagenous protein is selectively preserved in these tissues as a main protein. Type I and type III collagens were detected as major components by means of liquid chromatography-mass spectrometry analysis after digestion with trypsin. These results indicate that the triple helical collagen molecule, which is resistant to proteinase digestion, has been preserved in the soft tissues of these frozen mammoths for 40,000 years.

Lysyl Oxidase‐like 2 Targets Specific Lysine residues within the 7S dodecamer of Collagen IV

Lysyl oxidase like 2 (LOXL2) has emerged as an attractive target for the development of therapies for different forms of tissue fibrosis including liver, lung, kidney and cancer-associated fibrosis. LOXL2 is a copper dependent enzyme that catalyzes the oxidative deamination of lysine residues to form lysyl aldehydes (allysine) in collagens. Allysines spontaneously condense into lysyl-derived crosslinks which are important for the deposition of collagens in the extracellular matrix (ECM). In our previous studies, we have established that LOXL2 promotes the formation of lysine-derived crosslinks in the 7S dodecamer, a triple-helical crosslinking domain formed by the overlap of four collagen IV molecules. Although the specific location lysine residues targeted by lysyl oxidase have been described for fibrillar collagens, the equivalent information for 7S dodecamer of collagen IV is unknown. To better understand the mechanism by which LOXL2 mediates the crosslinking of collagen IV, we developed a fluorometric assay to detect amine oxidase activity using 7S dodecamer as substrate. We characterized the ability of LOXL2 to deaminate lysine residues in two forms of 7S substrate: the wild-type crosslinked 7S substrate and an uncrosslinked form purified from ECM derived from cells lacking LOXL2. Surprisingly, the enzyme showed little to no activity with crosslinked 7S dodecamer whereas a pronounced enzyme activity was observed with the uncrosslinked form of the 7S substrate. As trypsin digestion indicated that not all lysine residues present in the wild type 7S dodecamer take part in crosslinking, we hypothesized that the LOXL2 targets specific lysine residues. To identify specific lysine residues, uncrosslinked 7S dodecamers were oxidized with LOXL2 and newly formed allysines were labeled with a biotin tag. Optimization of biotinylation reaction was monitored by Streptavidin-HRP followed by enhanced chemiluminescence (ECL). After trypsin digestion of 7S dodecamers, biotinylated peptides containing allysine residues were identified by mass spectrometry. We discuss the potential rol of the identified allysines in the alignment of the four triple-helical molecules forming the 7S dodecamer. A better understanding of the mechanism by which LOXL2 promotes collagen IV crosslinking will provide the foundations for the development of effective therapies against tissue fibrosis.

Targeting cellular stress in vitro improves osteoblast homeostasis, matrix collagen content and mineralization in two murine models of osteogenesis imperfecta

Most cases of dominantly inherited osteogenesis imperfecta (OI) are caused by glycine substitutions in the triple helical domain of type I collagen α chains, which delay collagen folding, and cause the synthesis of collagen triple helical molecules with abnormal structure and post-translational modification. A variable extent of mutant collagen ER retention and other secondary mutation effects perturb osteoblast homeostasis and impair bone matrix quality. Amelioration of OI osteoblast homeostasis could be beneficial both to osteoblast anabolic activity and to the content of the extracellular matrix they deposit. Therefore, the effect of the chemical chaperone 4-phenylbutyrate (4-PBA) on cell homeostasis, collagen trafficking, matrix production and mineralization was investigated in primary osteoblasts from two murine models of moderate OI, Col1a1+/G349C and Col1a2+/G610C. At the cellular level, 4-PBA prevented intracellular accumulation of collagen and increased protein secretion, reducing aggregates within the mutant cells and normalizing ER morphology. At the extracellular level, increased collagen incorporation into matrix, associated with more mature collagen fibrils, was observed in osteoblasts from both models. 4-PBA also promoted OI osteoblast mineral deposition by increasing alkaline phosphatase expression and activity. Targeting osteoblast stress with 4-PBA improved both cellular and matrix abnormalities in culture, supporting further in vivo studies of its effect on bone tissue composition, strength and mineralization as a potential treatment for classical OI.

Ligand Exchange Strategy for Tuning of Helicity Inversion Speeds of Dynamic Helical Tri(saloph) Metallocryptands

AbstractInvited for the cover of this issue is the group of Shigehisa Akine at the Kanazawa University and the University of Tsukuba. The image depicts a scene with a triple helical molecule with a right‐handed helicity going into the world of mirror images upon the ligand exchange of the initial amines (orange‐colored) with the entering amines (green‐colored). Read the full text of the article at 10.1002/chem.201805799.

Type I collagen induces mesenchymal cell differentiation into myofibroblasts through YAP-induced TGF-β1 activation

In organ fibrosis, mechanical stress and transforming growth factor beta-1 (TGF-β1) promote differentiation into myofibroblast from mesenchymal cells, leading to extracellular matrix (ECM) remodeling or active synthesis, deposition or degradation of ECM components. A major component of ECM, type I collagen (col I) triple helical molecules assemble into fibrils or are denatured to gelatin without triple-helicity in remodeling. However, whether changes of ECM components in remodeling have influence on mesenchymal cell differentiation remains elusive. This study adopted three states of collagen I existing in ECM remodeling: molecular collagen, fibrillar collagen and gelatin to see what are characteristics in the effects on two cell lines of mesenchymal origin, murine 3T3-L1 embryonic fibroblast and murine C2C12 myoblasts. The results showed that all three forms of collagen I were capable of inducing these two cells to differentiate into myofibroblasts characterized by increased expression of alpha-smooth muscle actin (α-SMA) mRNA. The expression of α-SMA is positively regulated by TGF-β1. Nuclear translocation of Yes-associated protein (YAP) is involved in this process. Focal adhesion kinase (FAK) is activated in the cells cultured on molecular collagen-coated plates, contributing to YAP activation. On the other hand, in the cells cultured on fibrillar collagen gel or gelatin-coated plates, oxidative stress but not FAK induce YAP activation. In conclusion, the three physicochemically distinct forms of col I induce the differentiation of mesenchymal cells into myofibroblasts through different pathways.

Fatigue of soft fibrous tissues: Multi-scale mechanics and constitutive modeling

In recent experimental studies a possible damage mechanism of collagenous tissues mainly caused by fatigue was disclosed. In this contribution, a multi-scale constitutive model ranging from the tropocollagen (TC) molecule level up to bundles of collagen fibers is proposed and utilized to predict the elastic and inelastic long-term tissue response. Material failure of collagen fibrils is elucidated by a permanent opening of the triple helical collagen molecule conformation, triggered either by overstretching or reaction kinetics of non-covalent bonds. This kinetics is described within a probabilistic framework of adhesive detachments of molecular linkages providing collagen fiber integrity. Both intramolecular and interfibrillar linkages are considered. The final constitutive equations are validated against recent experimental data available in literature for both uniaxial tension to failure and the evolution of fatigue in subsequent loading cycles. All material parameters of the proposed model have a clear physical interpretation. Statement of significanceIrreversible changes take place at different length scales of soft fibrous tissues under supra-physiological loading and alter their macroscopic mechanical properties. Understanding the evolution of those histologic pathologies under loading and incorporating them into a continuum mechanical framework appears to be crucial in order to predict long-term evolution of various diseases and to support the development of tissue engineering.

Second-harmonic generation imaging of collagen in ancient bone

Second-harmonic generation imaging (SHG) captures triple helical collagen molecules near tissue surfaces. Biomedical research routinely utilizes various imaging software packages to quantify SHG signals for collagen content and distribution estimates in modern tissue samples including bone. For the first time using SHG, samples of modern, medieval, and ice age bones were imaged to test the applicability of SHG to ancient bone from a variety of ages, settings, and taxa. Four independent techniques including Raman spectroscopy, FTIR spectroscopy, radiocarbon dating protocols, and mass spectrometry-based protein sequencing, confirm the presence of protein, consistent with the hypothesis that SHG imaging detects ancient bone collagen. These results suggest that future studies have the potential to use SHG imaging to provide new insights into the composition of ancient bone, to characterize ancient bone disorders, to investigate collagen preservation within and between various taxa, and to monitor collagen decay regimes in different depositional environments.

The Path to Anatomy via Molecular Biology

Prior to the advent of Maxam and Gilbert DNA sequencing and Sanger, Nicklen and Coulson DNA sequencing, I was a student in an RNA sequencing laboratory. The experience taught me the proper paranoia for isolating and handling RNA. This was useful when, in the 1980s, I joined the lab of Bjorn Olsen at Rutgers Medical School, now called Robert Wood Johnson Medical School. There I learned the most current procedures in molecular biology, DNA sequencing and cloning. Bjorn was a great mentor, and he was an interesting guy. One day, another student and I found his histology notebook on his bookshelf‐‐he had hand drawn and colored every tissue section he had viewed so that he could study them for his histology exams. They were beautiful, accurate works of art, so intricate that I wished I could draw like that. Bjorn's passion about his work was contagious, and as a consequence, I fell in love with the family of collagens, which consisted of only 9 or 10 members then. A postdoc in the Olsen lab, Yoshifumi Ninomiya, had a role in determining the structure of type IX collagen by making a cDNA library from cartilage mRNAs and isolating the cDNAs corresponding to the collagen IX polypeptide chains. I was in awe of the idea that there were molecular biology strategies that could be used to identify and characterize new collagens, all because these triple helical molecules had repeating Gly‐Xxx‐Yyy sequences. The thrill of potentially being able to discover a new collagen made me love this type of science even more, and I put my whole heart into it. Around that time, a postdoc from Elizabeth Hay's lab visited Bjorn's lab, and this led to my meeting one of the best friends I'll ever have. That person, Kathy Svoboda, is not only partially responsible for Bjorn being hired by Betty Hay, chair of the Department of Anatomy and Cell Biology at Harvard Medical School, but her suggestion meant my life would change too! If Bjorn went to Boston, my husband Don and I would also get to go. And we did! It was an exciting experience to be in that department. Kathy, Betty, Bjorn and other faculty would talk about the American Association of Anatomists, and Kathy convinced me that AAA had investigators and educators that covered just about any topic in the biological sciences that could possibly interest me. She talked me into attending one of the annual meetings. I joined AAA sometime in the late 1980s or early 1990s, and, finding that what Kathy had said was true, I wanted to play a role in the society. I began serving on a committee as a guest member in 1999, and by 2001 I was committed to giving back to the Association since I had gotten so much from it. As I met more and more of the AAA members, I realized I now had greatly extended my “family” of scientists from this society, and that they played a big role in my being happy in my career.Support or Funding InformationSupported by National Eye Institute EY009056, National Institute of Arthritis and Musculoskeletal and Skin Diseases U54AR055073, and National Institute of Environmental Health Sciences P30ES005022

The binding capacity of α1β1-, α2β1- and α10β1-integrins depends on non-collagenous surface macromolecules rather than the collagens in cartilage fibrils

Interactions of cells with supramolecular aggregates of the extracellular matrix (ECM) are mediated, in part, by cell surface receptors of the integrin family. These are important molecular components of cell surface-suprastructures regulating cellular activities in general. A subfamily of β1-integrins with von Willebrand-factor A-like domains (I-domains) in their α-chains can bind to collagen molecules and, therefore, are considered as important cellular mechano-receptors. Here we show that chondrocytes strongly bind to cartilage collagens in the form of individual triple helical molecules but very weakly to fibrils formed by the same molecules. We also find that chondrocyte integrins α1β1-, α2β1- and α10β1-integrins and their I-domains have the same characteristics. Nevertheless we find integrin binding to mechanically generated cartilage fibril fragments, which also comprise peripheral non-collagenous material. We conclude that cell adhesion results from binding of integrin-containing adhesion suprastructures to the non-collagenous fibril periphery but not to the collagenous fibril cores. The biological importance of the well-investigated recognition of collagen molecules by integrins is unknown. Possible scenarios may include fibrillogenesis, fibril degradation and/or phagocytosis, recruitment of cells to remodeling sites, or molecular signaling across cytoplasmic membranes. In these circumstances, collagen molecules may lack a fibrillar organization. However, other processes requiring robust biomechanical functions, such as fibril organization in tissues, cell division, adhesion, or migration, do not involve direct integrin-collagen interactions.

Molecular assessment of collagen denaturation in decellularized tissues using a collagen hybridizing peptide.

Decellularized extracellular matrix (ECM) derived from tissues and organs are emerging as important scaffold materials for regenerative medicine. Many believe that preservation of the native ECM structure during decellularization is highly desirable. However, because effective techniques to assess the structural damage in ECM are lacking, the disruptive effects of a decellularization method and the impact of the associated structural damage upon the scaffold’s regenerative capacity are often debated. Using a novel collagen hybridizing peptide (CHP) that specifically binds to unfolded collagen chains, we investigated the molecular denaturation of collagen in the ECM decellularized by four commonly used cellremoving detergents: sodium dodecyl sulfate (SDS), 3-[(3-cholamidopropyl)dimethylammonio]-1-propa nesulfonate (CHAPS), sodium deoxycholate (SD), and Triton X-100. Staining of the detergent-treated porcine ligament and urinary bladder matrix with carboxyfluorescein-labeled CHP demonstrated that SDS and Triton X-100 denature the triple helical collagen molecule while CHAPS and SD do not, although second harmonic generation imaging and transmission electron microscopy (TEM) revealed that all four detergents disrupt collagen fibrils. Our findings from the CHP staining were further confirmed by the circular dichroism spectra of intact triple helical collagen molecules in CHAPS and SD solutions, and the TEM images of CHP-conjugated gold nanoparticles binding only to the SDS and Triton X-100 treated collagen fibrils. CHP is a powerful new tool for direct and reliable measurement of denatured collagen molecules in decellularized tissues. It is expected to have wide applications in the development and standardization of the tissue/organ decellularization technology.Statement of SignificancePreservation of the native ECM structure in decellularized tissues is highly desirable, since denaturation of ECM molecules (e.g., collagen) during decellularization can strongly influence the cellular response. Unfortunately, conventional techniques (SEM, SHG) are not conducive to identifying denatured collagen molecules in tissues. We demonstrate the first investigation into the molecular denaturation of collagen in decellularized ECM enabled by a novel Collagen Hybridizing Peptide (CHP) that specifically binds to unfolded collagen chains. We show that SDS and Triton X-100 denature collagen molecules while CHAPS and SD cannot. Such detection has been nearly impossible with other existing techniques. The CHP technique will advance our understanding about the effect of the cell-removing process on ECM, and lead to development of the decellularization technology.

Fell Muir Lecture: Collagen fibril formation in vitro and in vivo.

It is a great honour to be awarded the Fell Muir Prize for 2016 by the British Society of Matrix Biology. As recipient of the prize, I am taking the opportunity to write a minireview on collagen fibrillogenesis, which has been the focus of my research for 33years. This is the process by which triple helical collagen molecules assemble into centimetre-long fibrils in the extracellular matrix of animals. The fibrils appeared a billion years ago at the dawn of multicellular animal life as the primary scaffold for tissue morphogenesis. The fibrils occur in exquisite three-dimensional architectures that match the physical demands of tissues, for example orthogonal lattices in cornea, basket weaves in skin and blood vessels, and parallel bundles in tendon, ligament and nerves. The question of how collagen fibrils are formed was posed at the end of the nineteenth century. Since then, we have learned about the structure of DNA and the peptide bond, understood how plants capture the sun's energy, cloned animals, discovered antibiotics and found ways of editing our genome in the pursuit of new cures for diseases. However, how cells generate tissues from collagen fibrils remains one of the big unsolved mysteries in biology. In this review, I will give a personal account of the topic and highlight some of the approaches that my research group are taking to find new insights.

Lysyl Hydroxylase 2 Is Secreted by Tumor Cells and Can Modify Collagen in the Extracellular Space

Lysyl hydroxylase 2 (LH2) catalyzes the hydroxylation of lysine residues in the telopeptides of fibrillar collagens, which leads to the formation of stable collagen cross-links. Recently we reported that LH2 enhances the metastatic propensity of lung cancer by increasing the amount of stable hydroxylysine aldehyde-derived collagen cross-links (HLCCs), which generate a stiffer tumor stroma (Chen, Y., et al. (2015) J. Clin. Invest. 125, 125, 1147–1162). It is generally accepted that LH2 modifies procollagen α chains on the endoplasmic reticulum before the formation of triple helical procollagen molecules. Herein, we report that LH2 is also secreted and modifies collagen in the extracellular space. Analyses of lung cancer cell lines demonstrated that LH2 is present in the cell lysates and the conditioned media in a dimeric, active form in both compartments. LH2 co-localized with collagen fibrils in the extracellular space in human lung cancer specimens and in orthotopic lung tumors generated by injection of a LH2-expressing human lung cancer cell line into nude mice. LH2 depletion in MC3T3 osteoblastic cells impaired the formation of HLCCs, resulting in an increase in the unmodified lysine aldehyde-derived collagen cross-link (LCC), and the addition of recombinant LH2 to the media of LH2-deficient MC3T3 cells was sufficient to rescue HLCC formation in the extracellular matrix. The finding that LH2 modifies collagen in the extracellular space challenges the current view that LH2 functions solely on the endoplasmic reticulum and could also have important implications for cancer biology.

Comprehensive Characterization of Glycosylation and Hydroxylation of Basement Membrane Collagen IV by High-Resolution Mass Spectrometry.

Collagen IV is the main structural protein that provides a scaffold for assembly of basement membrane proteins. Posttranslational modifications such as hydroxylation of proline and lysine and glycosylation of lysine are essential for the functioning of collagen IV triple-helical molecules. These modifications are highly abundant posing a difficult challenge for in-depth characterization of collagen IV using conventional proteomics approaches. Herein, we implemented an integrated pipeline combining high-resolution mass spectrometry with different fragmentation techniques and an optimized bioinformatics workflow to study posttranslational modifications in mouse collagen IV. We achieved 82% sequence coverage for the α1 chain, mapping 39 glycosylated hydroxylysine, 148 4-hydroxyproline, and seven 3-hydroxyproline residues. Further, we employed our pipeline to map the modifications on human collagen IV and achieved 85% sequence coverage for the α1 chain, mapping 35 glycosylated hydroxylysine, 163 4-hydroxyproline, and 14 3-hydroxyproline residues. Although lysine glycosylation heterogeneity was observed in both mouse and human, 21 conserved sites were identified. Likewise, five 3-hydroxyproline residues were conserved between mouse and human, suggesting that these modification sites are important for collagen IV function. Collectively, these are the first comprehensive maps of hydroxylation and glycosylation sites in collagen IV, which lay the foundation for dissecting the key role of these modifications in health and disease.

The contribution of solid-state NMR spectroscopy to understanding biomineralization: Atomic and molecular structure of bone

Solid-state NMR spectroscopy has had a major impact on our understanding of the structure of mineralized tissues, in particular bone. Bone exemplifies the organic–inorganic composite structure inherent in mineralized tissues. The organic component of the extracellular matrix in bone is primarily composed of ordered fibrils of collagen triple-helical molecules, in which the inorganic component, calcium phosphate particles, composed of stacks of mineral platelets, are arranged around the fibrils. This perspective argues that key factors in our current structural model of bone mineral have come about through NMR spectroscopy and have yielded the primary information on how the mineral particles interface and bind with the underlying organic matrix. The structure of collagen within the organic matrix of bone or any other structural tissue has yet to be determined, but here too, this perspective shows there has been real progress made through application of solid-state NMR spectroscopy in conjunction with other techniques. In particular, NMR spectroscopy has highlighted the fact that even within these structural proteins, there is considerable dynamics, which suggests that one should be cautious when using inherently static structural models, such as those arising from X-ray diffraction analyses, to gain insight into molecular roles. It is clear that the NMR approach is still in its infancy in this area, and that we can expect many more developments in the future, particularly in understanding the molecular mechanisms of bone diseases and ageing.

Collagen in Health and Disease

Collagen is the unique, triple helical protein molecule which forms the major part of the extracellular matrix. It is the most abundant protein in the human body, representing 30% of its dry weight and is important to health because it characterizes the structure of skin, connective tissues, tendons, bones and cartilage. As collagen forms building block of body structures, any defect in collagen results in disorders, such as osteogenesis imperfecta, Ehlers-Dalnos syndrome, scurvy, systemic lupus erythematosus, systemic sclerosis, Stickler syndrome, oral submucous fibrosis, Marfan syndrome, epidermolysis bullosa, Alport syndrome. This review discusses the role of collagen in health as well as disease.

Structural and Mechanical Differences between Collagen Homo- and Heterotrimers: Relevance for the Molecular Origin of Brittle Bone Disease

Collagen constitutes one-third of the human proteome, providing mechanical stability, elasticity, and strength to organisms. Normal type I collagen is a heterotrimer triple-helical molecule consisting of two α-1 chains and one α-2 chain. The homotrimeric isoform of type I collagen, which consists of three α-1 chains, is only found in fetal tissues, fibrosis, and cancer in humans. A mouse model of the genetic brittle bone disease, osteogenesis imperfect, oim, is characterized by a replacement of the α-2 chain by an α-1 chain, resulting also in a homotrimer collagen molecule. Experimental studies of oim mice tendon and bone have shown reduced mechanical strength compared to normal mice. The relationship between the molecular content and the decrease in strength is, however, still unknown. Here, fully atomistic simulations of a section of mouse type I heterotrimer and homotrimer collagen molecules are developed to explore the effect of the substitution of the α-2 chain. We calculate the persistence length and carry out a detailed analysis of the structure to determine differences in structural and mechanical behavior between hetero- and homotrimers. The results show that homotrimer persistence length is half of that of the heterotrimer (96 Å vs. 215 Å), indicating it is more flexible and confirmed by direct mechanical testing. Our structural analyses reveal that in contrast to the heterotrimer, the homotrimer easily forms kinks and freely rotates with angles much larger than heterotrimer. These local kinks may explain the larger lateral distance between collagen molecules seen in the fibrils of oim mice tendon and could have implications for reducing the intermolecular cross-linking, which is known to reduce the mechanical strength.

Designed Coiled Coils Promote Folding of a Recombinant Bacterial Collagen

Collagen triple helices fold slowly and inefficiently, often requiring adjacent globular domains to assist this process. In the Streptococcus pyogenes collagen-like protein Scl2, a V domain predicted to be largely α-helical, occurs N-terminal to the collagen triple helix (CL). Here, we replace this natural trimerization domain with a de novo designed, hyperstable, parallel, three-stranded, α-helical coiled coil (CC), either at the N terminus (CC-CL) or the C terminus (CL-CC) of the collagen domain. CD spectra of the constructs are consistent with additivity of independently and fully folded CC and CL domains, and the proteins retain their distinctive thermal stabilities, CL at ∼37 °C and CC at >90 °C. Heating the hybrid proteins to 50 °C unfolds CL, leaving CC intact, and upon cooling, the rate of CL refolding is somewhat faster for CL-CC than for CC-CL. A construct with coiled coils on both ends, CC-CL-CC, retains the ∼37 °C thermal stability for CL but shows less triple helix at low temperature and less denaturation at 50 °C. Most strikingly however, in CC-CL-CC, the CL refolds slower than in either CC-CL or CL-CC by almost two orders of magnitude. We propose that a single CC promotes folding of the CL domain via nucleation and in-register growth from one end, whereas initiation and growth from both ends in CC-CL-CC results in mismatched registers that frustrate folding. Bioinformatics analysis of natural collagens lends support to this because, where present, there is generally only one coiled-coil domain close to the triple helix, and it is nearly always N-terminal to the collagen repeat.

Abstract 557: Single-chain type IV collagen (SCCOL-IV), an alternative gene product of COL4A1 as a therapeutic target in tumor tissue expansion as well as a diagnostic marker

Abstract Type IV collagen (COL-IV) is a major component of basement membrane (BM), comprising a polygonal meshwork structure underlying the basal side of polarized epithelia in normal tissues. Modulation of BM due to COL-IV degradation by matrix metalloproteinases is often associated with tumor aggravation and metastasis; Degradation products of BM components, including laminin and type IV collagen e.g., are involved in promoting tumor cell motility. Our previous studies showed that cultured human cells, in particular under ascorbate-deficiency, secrete single-chain COL-IV (SCCOL-IV), which is not derived from the denatured polypeptide of COL-IV triple-helical molecule. SCCOL-IV undergoes posttranslational modifications distinct from triple-helical COL-IV; SCCOL-IV has prolyl- and lysyl-hydroxylation to a lower extent than triple-helical COL-IV. SCCOL-IV contains the O-glycan structure of Galβ1-3GalNAc with a positive reaction to Agaricus bisporus agglutinin. The lectin does not recognize the triple-helical COL-IV-derived polypeptide. The alternative form of COL4A1 gene product is detected in vivo by the antibody that recognizes only SCCOL-IV. The polypeptide is isolated from human placenta as well as cultured media. In the present study, we demonstrate that SCCOL-IV is expressed specifically by cultured tumor cells and in tumor tissues. One of the antibodies against SCCOL-IV repressed tumor growth in an in vivo mouse model. The culture cell production of SCCOL-IV was demonstrated by Western blotting of cultured media, with an anti-SCCOL IV antibody, JK132 (Connective Tissue 31:161-168, 1999) for human cancer cell lines; Lu65A and NCI-H460 (pulmonary cancer), HLF (hepatoma), UO31 and A498 (renal cancer), and Panc-1 (pancreatic cancer). SCCOL-IV expression level was as high in tumor tissues of Lu65A-bearing nude mice as in tumor lesions of pulmonary cancer patients. One of the monoclonal antibodies against purified SCCOL-IV, NK46141, which exhibits a higher affinity compared with JK132, has no cross-reactivity with triple-helical COL-IV. Prophylactic interventions with NK46141 suppressed tumor growth in Lu65A-bearing nude mice. NK46141 had no effect, however, on the proliferation of cultured Lu65A cells. In order to obtain a clue for a mechanism how the antibody suppresses in vivo tumor growth, biological properties of SCCOL-IV in tumor interstitium were examined. SCCOL-IV was localized predominantly in vessel lumens in an in vitro vasculogenesis model, suggesting that SCCOL-IV may play a critical role for vasculogenesis in tumor progression. Prevention tumor growth by the antibody might be due to vasculogenesis inhibition. We propose that SCCOL-IV is a useful target in tumor diagnosis and therapy. Anti-SCCOL-IV antibodies including NK46141 could be potential therapeutics for tumor progression involving vasculogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 557. doi:10.1158/1538-7445.AM2011-557