Animal Extracellular Matrix Research Articles

Hydroxylation of recombinant human collagen type I alpha 1 in transgenic maize co-expressed with a recombinant human prolyl 4-hydroxylase

BackgroundCollagens require the hydroxylation of proline (Pro) residues in their triple-helical domain repeating sequence Xaa-Pro-Gly to function properly as a main structural component of the extracellular matrix in animals at physiologically relevant conditions. The regioselective proline hydroxylation is catalyzed by a specific prolyl 4-hydroxylase (P4H) as a posttranslational processing step.ResultsA recombinant human collagen type I α-1 (rCIα1) with high percentage of hydroxylated prolines (Hyp) was produced in transgenic maize seeds when co-expressed with both the α- and β- subunits of a recombinant human P4H (rP4H). Germ-specific expression of rCIα1 using maize globulin-1 gene promoter resulted in an average yield of 12 mg/kg seed for the full-length rCIα1 in seeds without co-expression of rP4H and 4 mg/kg seed for the rCIα1 (rCIα1-OH) in seeds with co-expression of rP4H. High-resolution mass spectrometry (HRMS) analysis revealed that nearly half of the collagenous repeating triplets in rCIα1 isolated from rP4H co-expressing maize line had the Pro residues changed to Hyp residues. The HRMS analysis determined the Hyp content of maize-derived rCIα1-OH as 18.11%, which is comparable to the Hyp level of yeast-derived rCIα1-OH (17.47%) and the native human CIa1 (14.59%), respectively. The increased Hyp percentage was correlated with a markedly enhanced thermal stability of maize-derived rCIα1-OH when compared to the non-hydroxylated rCIα1.ConclusionsThis work shows that maize has potential to produce adequately modified exogenous proteins with mammalian-like post-translational modifications that may be require for their use as pharmaceutical and industrial products.

Comparative Performance of Electrospun Collagen Nanofibers Cross-linked by Means of Different Methods

Collagen, as the major structural protein of the extracellular matrix in animals, is a versatile biomaterial of great interest in various engineering applications. Electrospun nanofibers of collagen are regarded as very promising materials for tissue engineering applications because they can reproduce the morphology of the natural bone but have as a drawback a poor structural consistency in wet conditions. In this paper, a comparative study between the performance of different cross-linking methods such as a milder enzymatic treatment procedure using transglutaminase, the use of N-[3-(dimethylamino)propyl]-N'-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide, and genipin, and the use of a physical method based on exposure to ultraviolet light was carried out. The chemical and enzymatic treatments provided, in this order, excellent consistency, morphology, cross-linking degree, and osteoblast viability for the collagen nanofibers. Interestingly, the enzymatically cross-linked collagen mats, which are considered to be a more biological treatment, promoted adequate cell adhesion, making the biomaterial biocompatible and with an adequate degree of porosity for cell seeding and in-growth.

Glycotoxins: a possible threat to health?

AGEs are produced by a nonenzymatic glycation process called the Maillard reaction which involves the condensation of a reducing sugar or an aldehydic group and a protein amino group, with the formation of a series of reactive intermediates leading to stable, irreversible, seldom fluorescent, compounds known as advanced glycation end-products (AGEs). The reaction occurs both in food during heating and in animal and human tissues. During the heating of food containing sugars, lipids and proteins, nonenzymatic protein browning reaction occurs, resulting in the formation of a large series of compounds, which include the melanoidins and other AGEs. Accumulation of AGEs in the cells and in the extracellular matrix in animals is also observed; it is age-dependent, is related to cell or tissue turnover and is species-specific. These processes are accelerated in chronic diseases in which the production of pathogenetic AGEs and their accumulation rate are increased. The two research fields, the Maillard reaction in food chemistry and the glycation cascade in medicine, ran in parallel independently for a long time, but recently a new scientific society (the International Maillard Reaction Society, IMARS) has opened the way for an exciting fusion of knowledge. Pioneering studies resulted in concerns about the non-negligible bioavailability of dietary AGEs and related plasma concentrations in humans, and there are now claims that dietary AGEs may be toxic because of their bioreactivity in different chronic diseases. This has been confirmed by several studies in animals and in humans (AGEs-restricted diets resulted in a reduction of serum and tissue AGEs, related AGE receptors, inflammatory cells, and proatherosclerotic factors) and by in vitro studies on cells in culture. In view of the emerging evidence of a pathogenetic role of glycotoxins, an assessment of the benefits of a shift to a specific nutritional approach (the Mediterranean diet being the healthiest in terms of the low amount of AGEs and high amount of antioxidants) and an innovative therapeutic approach, aimed at directly breaking AGEs molecules or reversing their impaired gut absorption to favour their renal or faecal excretion, may be warranted. At any rate, a new scientific field is open for an interdisciplinary debate (involving chemists, geriatricians, and specialists in metabolic disorders, diabetes, and diet) paving the way for possible, remarkable clinical progress.

The genome of the choanoflagellate Monosiga brevicollis and the origin of metazoans

Choanoflagellates are the closest known relatives of metazoans. To discover potential molecular mechanisms underlying the evolution of metazoan multicellularity, we sequenced and analysed the genome of the unicellular choanoflagellate Monosiga brevicollis. The genome contains approximately 9,200 intron-rich genes, including a number that encode cell adhesion and signalling protein domains that are otherwise restricted to metazoans. Here we show that the physical linkages among protein domains often differ between M. brevicollis and metazoans, suggesting that abundant domain shuffling followed the separation of the choanoflagellate and metazoan lineages. The completion of the M. brevicollis genome allows us to reconstruct with increasing resolution the genomic changes that accompanied the origin of metazoans.

Nanoarchitectures of the animal extracellular matrix: opportunities for synchrotron radiation studies on collagen and fibrillin

The extracellular matrix comprises structures that support the architectural organization of virtually all animal tissues. Within this architecture, two classes of protein assemblies found as long slender fibrils (collagen and fibrillin) characterize the bulk of the extracellular matrix. In both classes of fibrous protein, the molecular organization within a fibril ensures that the properties of the individual molecules transcend to the nanostructural and mesoscopic levels of structural organization and thence the tissue itself. The composition of the fibrils, in conjunction with other biomolecules and their suprafibrillar architecture, facilitates the formation of tissues as diverse as skin, tendon, cornea ciliary zonules and aorta. Here the relative tear resistance, strength, transparency and optical properties are paramount for proper function. Many structural investigations of fibrous protein structure have relied heavily on the use of synchrotron radiation in order to elucidate molecular packing, primarily due to the distinct benefits that X-ray diffraction provides, such as minimal sample preparation, rapid data collection and in situ mechanical testing. In this paper, an overview of the investigations that have revealed different levels of molecular architecture in fibril-based tissues is presented. Emerging future technology and how this can be matched with the pressing questions in extracellular matrix biology are also discussed.

Synthesis and Application of Gold Nanoparticles Functionalized with Collagen Mimetic Peptides

ABSTRACTCollagen is the principal tensile element of the extra-cellular matrix in animals and is the basic scaffold for cells and tissues. Abnormalities in its structure are known to result in a number of debilitating human diseases. Collagen mimetic peptides (CMPs) with repeat unit of (Pro-Hyp-Gly) are capable of forming right-handed triple-helical structures similar to that of the collagen triple helices. Recently, our group has shown that CMPs exhibit specific binding affinity to natural collagen under controlled thermal conditions. Using solid phase peptide synthesis, we have prepared a CMP cysteine derivative that was used to modify gold nanoparticles. Transmission electron microscopy (TEM) shows that the Cys-CMP functionalized gold nanoparticles have affinity to collagen fibers. We are investigating the interactions between Cys-CMP functionalized gold nanoparticles and collagen fibers. The Cys-CMP conjugated nanoparticles can potentially be used as a tool to visualize and understand unstable domains of collagen fibers which are related to a number of pathological conditions of extra cellular matrices.

The MUR3 Gene of Arabidopsis Encodes a Xyloglucan Galactosyltransferase That Is Evolutionarily Related to Animal Exostosins

Xyloglucans are the principal glycans that interlace cellulose microfibrils in most flowering plants. The mur3 mutant of Arabidopsis contains a severely altered structure of this polysaccharide because of the absence of a conserved alpha-L-fucosyl-(1-->2)-beta-D-galactosyl side chain and excessive galactosylation at an alternative xylose residue. Despite this severe structural alteration, mur3 plants were phenotypically normal and exhibited tensile strength in their inflorescence stems comparable to that of wild-type plants. The MUR3 gene was cloned positionally and shown to encode a xyloglucan galactosyltransferase that acts specifically on the third xylose residue within the XXXG core structure of xyloglucan. MUR3 belongs to a large family of type-II membrane proteins that is evolutionarily conserved among higher plants. The enzyme shows sequence similarities to the glucuronosyltransferase domain of exostosins, a class of animal glycosyltransferases that catalyze the synthesis of heparan sulfate, a glycosaminoglycan with numerous roles in cell differentiation and development. This finding suggests that components of the plant cell wall and of the animal extracellular matrix are synthesized by evolutionarily related enzymes even though the structures of the corresponding polysaccharides are entirely different from each other.

Effect of the tetrapeptide RGDS on somatic embryogenesis in Daucus carota.

The present work examines the requirement for RGD-binding sites, such as those in the plasma membrane protein integrin during somatic embryogenesis in carrot (Daucus carota L. cv. Scarlet nantes). It is possible to assay for this requirement by competitively inhibiting binding of the site to the natural RGD-containing ligand by adding an excess of synthetic RGDS. We found that treatment of carrot callus cultures with RGDS (2.7 mM) inhibited the development of a normal shoot pole in carrot somatic embryos. The structures that formed contained separate zones of highly cytoplasmic and vacuolate cells and no evidence of embryonic organization, although occasionally a root-like structure was observed. If the aspartic acid residue in the peptide was replaced by a chemically similar amino acid (glutamic acid), the resultant somatic embryos were indistinguishable from those developing in untreated cultures. These effects are similar to those reported in animal systems where the protein receptor involved has been identified as integrin. Our results are thus consistent with the conclusion that a binding site for a motif similar to the integrin-binding site in a variety of animal extracellular matrix proteins exists in plants and appears to be important in somatic embryo development in carrot.

Isolation of a 41-kDa protein with cell adhesion activity for animal tumor cells from the mushroom Hypsizigus marmoreus by affinity chromatography with type IV collagen immobilized on agarose.

A type IV collagen-binding protein of 41 kDa was isolated from the mushroom Hypsizigus marmoreus and the protein was designated as HM41. The Western blotting analysis with anti-HM41 antibodies demonstrated that HM41 was unrelated to HM23, which had been shown to have an affinity for type IV collagen. The microsequence analysis of the membrane-blotted peptides generated by fragmentation with cyanogen bromide showed no homologous proteins reported. HM41 had cell adhesion-promoting activity for murine Lewis lung carcinoma LL2 cells and human fibrosarcoma HT1080 cells. These results indicate that HM41 is a hitherto undescribed fungus protein that can interact both with animal extracellular matrix protein type IV collagen and with animal tumor cells.

Cell adhesion activity for murine carcinoma cells of a wheat germ 55-kDa protein with binding affinity for animal extracellular matrix proteins

A wheat germ 55-kDa protein was isolated by affinity chromatography with Matrigel immobilized on agarose, followed by preparative gel electrophoresis. This Matrigel-binding protein designated as WG-55 had an amino-terminal amino acid sequence which is identical to that of a putative mature form of wheat storage protein Gbl 1. WG-55 reacted with concanavalin A, indicating its glycoprotein nature as expected from the amino acid sequence of Gbl 1. As expected, similarly, WG-55 exhibited RGD-dependent cell adhesion activity for murine carcinoma cells. These data suggest that WG-55 or mature Gbl 1 protein may play a role in plant cell adhesion.

Effect of crosslinking method on collagen fiber-fibroblast interactions

Collagen, the major structural protein of the extracellular matrix in animals, is a versatile biomaterial used in various tissue engineering applications. Cross-linking influences the mechanical properties, resorption kinetics, and biocompatibility of collagen-based biomaterials. In this study, we evaluated the effects of crosslinking on collagen fiber-fibroblast interactions in vitro. Collagen fibers were left untreated or crosslinked by ultraviolet (UV) irradiation, dehydrothermal (DHT) treatment (3 or 5 days), or hexamethylenediisocyanate (HMDIC) exposure. The initial attachment, proliferation (through 8 days), and morphology of human dermal fibroblasts were evaluated on control and crosslinked bundles of 200 collagen fibers in vitro. Initial attachment (number of fibroblasts at day 0) was increased on UV and DHT5-treated collagen fiber bundles. Fibroblast proliferation was similar for control, UV crosslinked, and DHT crosslinked fibers. In contrast, fibroblast attachment was significantly decreased and proliferation was delayed on HMDIC crosslinked fibers. These results, coupled with our previous studies, suggest that UV crosslinking of collagen fibers provides a combination of biocompatibility, mechanical properties, and strength retention suitable for various tissue engineering applications. © 1997 John Wiley & Sons, Inc. J Appl Polym Sci 63: 1493–1498, 1997

Immunolocalization of fimbrial epitopes in thin sections of Microbotryum violaceum

Fungal fimbriae are long (0.5–20 μm), narrow (7 nm) surface appendages that have been observed on most members of the Mycota. Biochemical analyses have determined that fimbriae from Microbotryum violaceum are composed of 74-kDa glycoproteinaceous subunits in which the protein moiety is fungal collagen. We present evidence for the localization of fimbrial subunits prior to their exportation from the cell. We term these internal, likely nonpolymerized fimbriae "pro-fimbriae" and demonstrate the location of the reserves within the peripheral cytoplasm. Also, we show that fimbriae may not traverse the cell wall as previously believed, but may instead originate from within the outer lamella of the cell wall, possibly being anchored to the cell wall via other molecules. This model is analogous to the animal extracellular matrix arrangement in which collagens are anchored to plasma membranes via other proteins such as fibronectin.Key words: fungus, immunolocalization, fimbriae, Microbotryum, Ustilago.

Fungal fimbriae are composed of collagen.

Fungal fimbriae are surface appendages that were first described on the haploid cells of the smut fungus, Microbotryum violaceum. They are long (1-20 microm), narrow (7 nm) flexuous structures that have been implicated in cellular functions such as mating and pathogenesis. Since the initial description, numerous fungi from all five phyla have been shown to produce fimbriae on their extracellular surfaces. The present study analyses the protein component of M.violaceum fimbriae. The N-terminus and three internal amino acid sequences were determined. All four show a strong similarity to sequences which are characteristic of the collagen gene family. Enzymatic digests and immunochemical analyses support this finding. Based on these results, it is suggested that the proteinaceous subunits of fimbriae should be termed fungal collagens. Previously, collagen has been found only among members of the kingdom Animalia where it is the principal component of the animal extracellular matrix and is the most abundant animal protein. The unexpected finding of collagen in the members of the Mycota suggests that it may have evolved from a common ancestor that existed before the divergence of fungi and animals. Further, native fungal fimbriae can function as a mammalian extracellular matrix component. They can act as a substratum which permits animal cells to adhere, spread, and proliferate in a manner similar to animal collagens. The implications of this finding to both phylogeny and pathology are discussed.

Isolation of a Novel Collagen-binding Protein from the Mushroom, Hypsizigus marmoreus, Which Inhibits the Lewis Lung Carcinoma Cell Adhesion to Type IV Collagen

A type IV collagen-binding protein of 23 kDa was isolated from the mushroom, Hypsizigus marmoreus. This protein, HM 23, bound to type IV and type I collagens and gelatin, and to much lesser extent to fibronectin, but not to laminin or bovine serum albumin. The adhesion of Lewis lung carcinoma cells was inhibited when the type IV collagen substratum was pretreated with HM 23. A computer search of the determined partial amino acid sequence indicated no homologous proteins reported. These results indicate that HM 23 is a hitherto undescribed fungus protein that can interact with animal extracellular matrix proteins.

Enrichment of vitronectin‐ and fibronectin‐like proteins in NaCl‐adapted plant cells and evidence for their involvement in plasma membrane–cell wall adhesion

Cells of tobacco adapted to grow in high concentrations of NaCl develop tight zones of adhesion between the plasma membrane and cell wall, revealed by concave plasmolysis in osmotic solutions. Unadapted cells exhibit mostly convex plasmolysis and exhibit little or no adhesive character. Wall-less protoplasts isolated from the adapted cells retain the complementary adhesive character and adhere tightly to each other, whereas protoplasts from unadapted cells do not. The hexapeptide gly-arg-gly-asp-ser-pro, in which the arg-gly-asp represents the integrin-binding domain of several animal extracellular matrix proteins,specifically blocks adhesion of the protoplasts. A control hexapeptide, gly-arg-gly-glu-ser-pro, is ineffective in blocking adhesion. Tobacco proteins immunologically related to human vitronectin were found in cell walls and membranes of unadapted and NaCI adapted cells, but the total extractable vitronectin-like protein was enriched in the adapted cells. Tobacco proteins immunologically related to human fibronectin were found in membranes and cell walls of NaCI adapted cells but not in those from unadapted cells.Our observations indicate that plant cells possess cell-matrix adhesion complexes similar to animal cells, and these adhesion complexes accumulate ingrowth-limited cells adapted to saline stress.

The effect of estradiol on collagen structure and organization in the immature rat uterus.

An examination of collagen ultrastructure in the lamina propria of the immature ovariectomized rat uterus revealed that a single injection of estradiol (40 micrograms/kg) produced a biphasic effect on collagen structure and organization. In saline-treated animals (controls) dense populations of collagen bundles were seen throughout the extracellular matrix (EX). Cross-sections of these bundles suggested that the bundles run parallel to the long axis of the uterus and thin filaments seemed to form cross-links between collagen fibers. In contrast, large clear spaces, collagen fragments, and loosely packed bundles of collagen were observed in the EX of animals injected with estradiol 24 hr earlier. In a time course study (0, 1, 2, 4, 24, and 48 hr), estradiol treatment altered collagen structure and organization 1 hr following administration. Collagen bundles did not appear to be as densely packed as in control tissues and large clear spaces were evident in the EX. Two hours following estrogen administration, collagen fibers appeared to be fragmented and seemed to be separating from the plasma membrane of stromal cells. Four hours following estrogen administration, large clear spaces occupied most of the EX in the lamina propria. Collagen fragments were diffusely distributed throughout the EX and small cross-sectional patches of collagen bundles were present. In 48-hr-treated animals, collagen bundles reappeared and were often closely associated with the plasma membrane of stromal cells. The collagen was not as abundant as in control animals. An overview of the cellular organization of the lamina propria revealed that stromal cells in control tissues were more densely packed than in estradiol-treated tissues (4, 24, and 48 hr) and the stromal cell size appeared to increase in hormone-treated tissues. These responses provide a good model system to study the role of estradiol in the control of collagen structure and organization in the uterus.