Cell attachment properties of collagen type VI and Arg-Gly-Asp dependent binding to its alpha 2(VI) and alpha 3(VI) chains

Twelve of sixteen different cell types including fibroblasts and tumor cells were able to attach and spread on substrates of pepsin-solubilized or intact collagen VI, and on its triple helical domain. Attachment and spreading were independent of soluble mediator proteins (fibronectin, laminin) and collagen VI was distinct from collagens I, IV and V in the cells with which it interacted. Many of the same cells bound and spread on substrates prepared from unfolded alpha 2(VI) and alpha 3(VI) chains but not on the alpha 1(VI) chain. The interactions with the chains were inhibited by low concentrations (10-100 microM) of synthetic RGDS and RGDT but not RGES peptides while the binding of cells to pepsin-solubilized collagen VI was more than 20-fold less sensitive to these peptides. The data indicate that cells have the ability to bind to collagen VI in a specific manner suggesting a similar function for collagen VI in situ.     

Biosynthesis of α2(IV) and α1(IV) chains of collagen IV and interactions with matrix metalloproteinase-9

In vitro binding studies with latent matrix metalloproteinase-9 (pro-MMP-9) have revealed the existence of nondisulfide-bonded α2(IV) chains on the cell surface capable of forming a high-affinity complex with the enzyme. Here we investigated the biosynthesis and cellular distribution of α2(IV) and α1(IV) chains in breast epithelial (MCF10A and MDA-MB-231) and fibrosarcoma (HT1080) cells by pulse-chase analysis followed by immunoprecipitation with chain-specific monoclonal antibodies (mAb). These studies showed that whereas the α1(IV) chain remained in the intracellular compartment, nondisulfide-bonded α2(IV) chains were secreted into the media in a stable form. Consistently, only α2(IV) was detected on the cell surface by surface biotinylation or indirect immunofluorescence. In agreement with the pulse-chase analysis, media subjected to coprecipitation experiments with pro-MMP-9 or pro-MMP-9-affinity chromatography followed by immunoblotting with chain-specific mAbs resulted in the detection of α2(IV). A preferential secretion of nondisulfide-bonded α2(IV) chains was also observed in CHO-K1 cells transiently transfected with full-length mouse α2(IV) or α1(IV) cDNAs. However, a complex of mouse α1(IV) with pro-MMP-9 was coprecipitated with exogenous enzyme from lysates of CHO-K1 cells transfected with mouse α1(IV), suggesting that under overexpression conditions the enzyme can also interact with the α1(IV) chain. Collectively, these studies further demonstrate the interactions of pro-MMP-9 with collagen IV chains and a unique processing and targeting of nondisulfide-bonded α2(IV) chains that may play a role in the surface/matrix association of pro-MMP-9. J. Cell. Physiol. 180:131–139, 1999. © 1999 Wiley-Liss, Inc.     

Role of the I-domain in collagen binding specificity and activation of the integrins α1β1 and α2β1

Adhesion to collagens by most cell types is mediated by the integrins α1β1 and α2β1. Both integrin α subunits belong to a group which is characterized by the presence of an I domain in the N-terminal half of the molecule, and this domain has been implicated in the ligand recognition. Since purified α1β1 and α2β1 differ in their binding to collagens I and IV and recognize different sites within the major cell binding domain of collagen IV, we investigated the potential role of the α1 and α2 I domains in specific collagen adhesion. We find that introducing the α2 I domain into α1 results in surface expression of a functional collagen receptor. The adhesion mediated by this chimeric receptor (α1-2-1β1) is similar to the adhesion profile conferred by α2β1, not α1β1. The presence of α2 or α1-2-1 results in preferential binding to collagen I, whereas α1 expressing cells bind better to collagen IV. In addition, α1 containing cells bind to low amounts of a tryptic fragment of collagen IV, whereas α2 or α1-2-1 bearing cells adhere only to high concentrations of this substrate. We also find that collagen adhesion of NIH-3T3 mediated by α2β1 or α1-2-1β1, but not by α1, requires the presence of Mn²⁺ ions. This ion requirement was not found in CHO cells, implicating the I domain in cell type-specific activation of integrins. J. Cell. Physiol. 176:634–641, 1998. © 1998 Wiley-Liss, Inc.     

Physical properties of type i collagen in solution: Structure of α-Chains and β- and γ-components and two-component mixtures

The structure of thermally denatured Type I collagen has been studied using laser light scattering. The results indicate that the diffusion coefficients of α-chains and β- and γ-components are 1.550 ± 0.08 × 10^?7, 1.000 ± 0.05 × 10^?7, and 0.835 ± 0.04 × 10^?7 cm²/sec, respectively, at temperatures between 20 and 40°C. It is concluded from diffusion data that these species have hydrodynamic radii of about 13.8 nm (α-chain), 21.5 nm (β-component), and 25.7 nm (γ-component), consistent with previous studies of thermal denaturation by light scattering. It is also concluded, based on volume calculations, that a large volume increase occurs when the triple helix unfolds. Homodyne correlation functions for two component mixtures of α-chains and β-and γ-components appeared to decay exponentially. In all but one case discussed the correlation function could be fitted with a single component having a translational diffusion coefficient which was an intensity weighted average of the diffusion coefficient of each component present.     

Packing constraints of hydrophobic side chains in (α/β)8 barrels

An analysis of possible tight packing of hydrophobic groups simultaneously at the both surfaces of β-hyperboloid-8 was conducted. This analysis shows that the disposition of amino acid side chains at the real β-structure's surface is unique. If we sign the mean distance between adjacent β-strands as “a,” and the mean distance along β-strand between C_α atoms, whose side chains are directed to one side of the β-sheet, as “b,” the ratio b/a = √2 very precisely. This ratio ensures the most efficient packing of side hydrophobic groups at the outer surface of β-hyperboloid-8, forming, at the same time, the second by efficiency packing at its inner surface. © 1995 Wiley-Liss, Inc.     

Molecular determinants of desensitization and assembly of the chimeric GABAA receptor subunits (α1/γ2) and (γ2/α1) in combinations with β2 and γ2

Two γ-aminobutyric acid_A (GABA_A) receptor chimeras were designed in order to elucidate the structural requirements for GABA_A receptor desensitization and assembly. The (α1/γ2) and (γ2/α1) chimeric subunits representing the extracellular N-terminal domain of α1 or γ2 and the remainder of the γ2 or α1 subunits, respectively, were expressed with β2 and β2γ2 in Spodoptera frugiperda (Sf-9) cells using the baculovirus expression system. The (α1/γ2)β2 and (α1/γ2)β2γ2 but not the (γ2/α1)β2 and (γ2/α1)β2γ2 subunit combinations formed functional receptor complexes as shown by whole-cell patch–clamp recordings and [³H]muscimol and [³H]flunitrazepam binding. Moreover, the surface immunofluorescence staining of Sf-9 cells expressing the (α1/γ2)-containing receptors was pronounced, as opposed to the staining of the (γ2/α1)-containing receptors, which was only slightly higher than background. To explain this, the (α1/γ2) and (γ2/α1) chimeras may act like α1 and γ2 subunits, respectively, indicating that the extracellular N-terminal segment is important for assembly. However, the (α1/γ2) chimeric subunit had characteristics different from the α1 subunit, since the (α1/γ2) chimera gave rise to no desensitization after GABA stimulation in whole-cell patch–clamp recordings, which was independent of whether the chimera was expressed in combination with β2 or β2γ2. Surprisingly, the (α1/γ2)(γ2/α1)β2 subunit combination did desensitize, indicating that the C-terminal segment of the α1 subunit may be important for desensitization. Moreover, desensitization was observed for the (α1/γ2)β2γ2 receptor with respect to the direct activation by pentobarbital. This suggests differences in the mechanism of channel activation for pentobarbital and GABA.     

Blood Platelets Contain and Secrete Laminin-8 (α4β1γ1) and Adhere to Laminin-8 via α6β1 Integrin

Laminins, a family of heterotrimeric proteins with cell adhesive/signaling properties, are characteristic components of basement membranes of vasculature and tissues. In the present study, permeabilized platelets were found to react with a monoclonal antibody to laminin γ1 chain by immunofluorescence. In Western blot analysis of platelet lysates, several monoclonal antibodies to γ1 and β1 laminin chains recognized 220- to 230-kDa polypeptides, under reducing conditions, and a structure with much slower electrophoretic mobility under nonreducing conditions. Immunoaffinity purification on a laminin β1 antibody–Sepharose column yielded polypeptides of 230, 220, 200, and 180 kDa from platelet lysates. In the purified material, mAbs to β1 and γ1 reacted with the two larger polypeptides, while affinity-purified rabbit antibodies to laminin α4 chain recognized the smallest polypeptide. Identity of the polypeptides was confirmed by microsequencing. One million platelets contained on average 1 ng of laminin (approximately 700 molecules per cell), of which 20–35% was secreted within minutes after stimulation with either thrombin or phorbol ester. Platelets adhered to plastic surfaces coated with the purified platelet laminin, and this process was largely inhibited by antibodies to β1 and α6 integrin chains. We conclude that platelets contain and, following activation, secrete laminin-8 (α4β1γ1) and that the cells adhere to the protein by using α6β1 integrin.     

The expression of α2β1 integrin and α smooth muscle actin in fibroblasts grown on collagen

The maturation of connective tissue involves the organization of collagen fibres by resident fibroblasts. Fibroblast attachment to collagen has been demonstrated to involve cell surface receptors, integrins of the β₁ family. Integrins are associated with cytoplasmic actin of microfilaments either directly or through focal adhesions. The major actin isoform of fibroblast microfilaments is β actin and to a lesser extent α smooth muscle (α SM) actin. Cultured human dermal fibroblasts derived from adult dermis, newborn foreskin or keloid scar were grown on either uncoated or collagen-coated surfaces. The expression and synthesis of both α₂β₁ integrin and α SM actin were followed by immunohistology and immunoprecipitation. Fibroblasts on uncoated surfaces expressed little α₂β₁ integrin on their surface, while 20 per cent of them demonstrated α SM actin within microfilaments. Fibroblasts grown on a collagen-coated surface minimally expressed α SM actin in microfilament structures and a majority of the cells were positive for α₂β₁ integrin on their membranes. Using [³⁵S]-methionine incorporation and immunoprecipitation, it was shown that fibroblasts grown in uncoated dishes synthesized more α SM actin than fibroblasts grown on collagen-coated dishes. In contrast, fibroblasts grown on collagen coated dishes synthesized more α₂β₁ integrin compared to the same cells grown on uncoated dishes. Fibroblasts maintained on a type I collagen upregulate the expression and synthesis of α₂β₁ integrin, and downregulate the expression and synthesis of α SM actin. © 1998 John Wiley & Sons, Ltd.     

Syndecan-1 and -4 differentially regulate oncogenic K-ras dependent cell invasion into collagen through α2β1 integrin and MT1-MMP

Syndecans function as co-receptors for integrins on different matrixes. Recently, syndecan-1 has been shown to be important for α2β1 integrin-mediated adhesion to collagen in tumor cells by regulating cell adhesion and migration on two-dimensional collagen. However, the function of syndecans in supporting α2β1 integrin interactions with three-dimensional (3D) collagen is less well studied. Using loss-of-function and overexpression experiments we show that in 3D collagen syndecan-4 supports α2β1-mediated collagen matrix contraction. Cell invasion through type I collagen containing 3D extracellular matrix (ECM) is driven by α2β1 integrin and membrane type-1 matrix metalloproteinase (MT1-MMP). Here we show that mutational activation of K-ras correlates with increased expression of α2β1 integrin, MT1-MMP, syndecan-1, and syndecan-4. While K-ras-induced α2β1 integrin and MT1-MMP are positive regulators of invasion, silencing and overexpression of syndecans demonstrate that these proteins inhibit cell invasion into collagen. Taken together, these data demonstrate the existence of a complex interplay between integrin α2β1, MT1-MMP, and syndecans in the invasion of K-ras mutant cells in 3D collagen that may represent a mechanism by which tumor cells become more invasive and metastatic.     

Structural classification of αββ and ββα supersecondary structure units in proteins

We present a fully automatic structural classification of supersecondary structure units, consisting of two hydrogen-bonded β strands, preceded or followed by an α helix. The classification is performed on the spatial arrangement of the secondary structure elements, irrespective of the length and conformation of the intervening loops. The similarity of the arrangements is estimated by a structure alignment procedure that uses as similarity measure the root mean square deviation of superimposed backbone atoms. Applied to a set of 141 well-resolved nonhomologous protein structures, the classification yields 11 families of recurrent arrangements. In addition, fragments that are structurally intermediate between the families are found; they reveal the continuity of the classification. The analysis of the families shows that the α helix and β hairpin axes can adopt virtually all relative orientations, with, however, some preferable orientations; moreover, according to the orientation, preferences in the left/right handedness of the α–β connection are observed. These preferences can be explained by favorable side by side packing of the α helix and the β hairpin, local interactions in the region of the α–β connection or stabilizing environments in the parent protein. Furthermore, fold recognition procedures and structure prediction algorithms coupled to database-derived potentials suggest that the preferable nature of these arrangements does not imply their intrinsic stability. They usually accommodate a large number of sequences, of which only a subset is predicted to stabilize the motif. The motifs predicted as stable could correspond to nuclei formed at the very beginning of the folding process. Proteins 30:193–212, 1998. © 1998 Wiley-Liss, Inc.     

Calorimetric studies of p-nitrophenol binding to α- and β-cyclodextrin

The thermodynamics of the binding of p-nitrophenol to both α- and β-cyclodextrin has been investigated as a function of pH and solvent composition using the technique of flow microcalorimetry. A preferential binding of the anionic form of the ligand is found for both cyclodextrins. It is postulated that the additional affinity for p-nitrophenolate is due to a dispersion interaction between the cyclodextrin cavity and the delocalized charge of the ligand. Studies of the binding of p-nitrophenol analogs and of the binding of p-nitrophenol as a function of ionic strength and DMSO cosolvent composition are consistent with this theory.     

α11β1 integrin‐mediated MMP‐13‐dependent collagen lattice contraction by fibroblasts: Evidence for integrin‐coordinated collagen proteolysis

We have previously determined that integrin α11β1 is required on mouse periodontal ligament (PDL) fibroblasts to generate the force needed for incisor eruption. As part of the phenotype of α11^?/? mice, the incisor PDL (iPDL) is thickened, due to disturbed matrix remodeling. To determine the molecular mechanism behind the disturbed matrix dynamics in the PDL we crossed α11^?/? mice with the Immortomouse and isolated immortalized iPDL cells. Microarray analysis of iPDL cells cultured inside a 3D collagen gel demonstrated downregulated expression of a number of genes in α11‐deficient iPDL cells, including matrix metalloproteinase‐13 (MMP‐13) and cathepsin K. α11^?/? iPDL cells in vitro displayed disturbed interactions with collagen I during contraction of attached and floating collagen lattices and furthermore displayed reduced MMP‐13 protein expression levels. The MMP‐13 specific inhibitor WAY 170523 and the Cathepsin K Inhibitor II both blocked part of the α11 integrin‐mediated collagen remodeling. In summary, our data demonstrate that in iPDL fibroblasts the mechanical strain generated by α11β1 integrin regulates molecules involved in collagen matrix dynamics. The positive regulation of α11β1‐dependent matrix remodeling, involving MMP‐13 and cathepsin K, might also occur in other types of fibroblasts and be an important regulatory mechanism for coordinated extracellular and intracellular collagen turnover in tissue homeostasis. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.     

2-(3′-Hydroxypropylidene)-1α-hydroxy-19-norvitamin D compounds with truncated side chains

Our recent studies with 2-(3′-hydroxypropylidene) analogs of 1α,25-dihydroxy-19-norvitamin D₃ showed that this 2-substituent creates compounds with very potent biological activity. In the continuing search for vitamin D compounds with selective activity profiles, we prepared a series of 1α-hydroxy-19-norvitamin D analogs characterized by the presence of a 3′-hydroxypropylidene substituent at C-2 and a truncated side chain. These vitamin D compounds were efficiently prepared using convergent syntheses. The C,D-fragments, namely the Grundmann ketones 19, 20, 27, 36 and 37 were synthesized from the known 8β-benzoyloxy-22-aldehydes 12 and 29. These hydrindanones were subjected to Lythgoe type Wittig–Horner coupling with phosphine oxide 21, prepared by us previously, and after hydroxyl deprotection the set of 19-norvitamins 7–11 was successfully obtained. According to our expectations, all analogs (with an exception of the 20R-compound 7) have pronounced in vitro activity. When compared to the natural hormone 1α,25-(OH)₂D₃ (1), they show the same or only slightly reduced affinity for the vitamin D receptor while being similarly effective as 1 in differentiation of HL-60 cells into monocytes.     

Transfection of normal human skin fibroblasts with human α1(I) and α2(I) collagen gene constructs and evidence for their coordinate expression

Type I collagen is composed of two α1(I) chains and one α2(I) chain that together form a unique triple helical structure. The genes for these chains are located on different chromosomes but their expression is tightly regulated. In order to investigate the mechanism of regulation of coordinate expression of these genes, I examined conditions for the efficient transfection of normal human skin fibroblasts with luciferase reporter gene constructs containing noncoding region of the first exon and the upper 500 base pairs sequence of the α1(I) or α2(I) gene. Expression ratio of these two reporter gene constructs was two to one, indicating these regions of α1(I) and α2(I) genes contain essential regulatory elements for the coordinate expression of α1(I) and α2(I) genes located on different chromosomes.     

Anti-inflammatory properties of α- and γ-tocopherol

Natural vitamin E consists of four different tocopherol and four different tocotrienol homologues (α, β, γ, δ) that all have antioxidant activity. However, recent data indicate that the different vitamin E homologues also have biological activity unrelated to their antioxidant activity. In this review, we discuss the anti-inflammatory properties of the two major forms of vitamin E, α-tocopherol (αT) and γ-tocopherol (γT), and discuss the potential molecular mechanisms involved in these effects. While both tocopherols exhibit anti-inflammatory activity in vitro and in vivo, supplementation with mixed (γT-enriched) tocopherols seems to be more potent than supplementation with αT alone. This may explain the mostly negative outcomes of the recent large-scale interventional chronic disease prevention trials with αT only and thus warrants further investigation.     

Determination of β-glucosidase aggregating factor (BGAF) binding and polymerization regions on the maize β-glucosidase isozyme Glu1

β-Glucosidases (Glu1 and Glu2) in maize specifically interact with a lectin called β-glucosidase aggregating factor (BGAF). We have shown that the N-terminal (Glu⁵⁰–Val¹⁴⁵) and the C-terminal (Phe⁴⁶⁶–Ala⁵¹²) regions of maize Glu1 are involved in binding to BGAF. Sequence comparison between sorghum β-glucosidases (dhurrinases, which do not bind to BGAF) and maize β-glucosidases, and the 3D-structure of Glu1 suggested that the BGAF-binding site on Glu1 is much smaller than predicted previously. To define more precisely the BGAF-binding site, we constructed additional chimeric β-glucosidases. The results showed that a region spanning 11 amino acids (Ile⁷²–Thr⁸²) on Glu1 is essential and sufficient for BGAF binding, whereas the extreme N-terminal region Ser¹–Thr²⁹, together with C-terminal region Phe⁴⁶⁶–Ala⁵¹², affects the size of Glu1–BGAF complexes. The dissociation constants (K_d) of chimeric β-glucosidase–BGAF interactions also demonstrated that the extreme N-terminal and C-terminal regions are important but not essential for binding. To confirm the importance of Ile⁷²–Thr⁸² on Glu1 for BGAF binding, we constructed a chimeric sorghum β-glucosidase, Dhr2 (C-11, Dhr2 whose Val⁷²–Glu⁸² region was replaced with the Ile⁷²–Thr⁸² region of Glu1). C-11 binds to BGAF, indicating that the Ile⁷²–Thr⁸² region is indeed a major interaction site on Glu1 involved in BGAF binding.     

Relationships between translation of pro α1(I) and pro α2(I) mRNAs during synthesis of the type I procollagen heterotrimer

Final assembly of the procollagen I heterotrimeric molecule is initiated by interactions between the carboxyl propeptide domains of completed, or nearly completed nascent pro α chains. These interactions register the chains for triple helix folding. Prior to these events, however, the appropriate nascent chains must be brought within the same compartments of the endoplasmic reticulum (ER). We hypothesize that the co-localization of the synthesis of the nascent pro α1(I) and pro α2(I) chains results from an interaction between their translational complexes during chain synthesis. This has been investigated by studying the polyribosomal loading of the pro α-chain messages during in vitro translation in the presence and absence of microsomal membranes, and in cells which have the ability to synthesize the pro α1 homotrimer or the normal heterotrimer. Recombinant human pro α1(I) and pro α2(I) C DNAs were inserted into plasmids and then transcribed in vitro. The resulting RNAs were translated separately and in mixture in a cell-free rabbit reticulocyte lysate ± canine pancreatic microsomes. Cycloheximide (100 μg/ml) was added and the polysomes were collected and fractionated on a 15–50% sucrose gradient. The RNA was extracted from each fraction and the level of each chain message was determined by RT-PCR. Polysomes from K16 (heterotrimer-producing), W8 (pro α1(I) homotrimer), and A2′ (heterotrimer + homotrimer) cells were similarly analyzed. Translations of the pro α1(I) and pro α2(I) messages proceeded independently in the cell-free, membrane-free systems, but were coordinately altered in the presence of membrane. The cell-free + membrane translation systems mimicked the behavior of the comparable cell polysome mRNA loading distributions. These data all suggest that there is an interaction between the pro α chain translational complexes at the ER membrane surface which temporally and spatially localize the nascent chains for efficient heteromeric selection and folding. © 1995 Wiley-Liss, Inc.     

Parallel solid-phase synthesis of 3β-peptido-3α-hydroxy-5α-androstan-17-one derivatives for inhibition of type 3 17β-hydroxysteroid dehydrogenase

Type 3 17β-hydroxysteroid dehydrogenase (17β-HSD), a key steroidogenic enzyme, transforms 4-androstene-3,17-dione (Δ⁴-dione) into testosterone. In order to produce potential inhibitors, we performed solid-phase synthesis of model libraries of 3β-peptido-3α-hydroxy-5α-androstan-17-ones with 1, 2, or 3 levels of molecular diversity, obtaining good overall yields (23–58%) and a high average purity (86%, without any purification steps) using the Leznoff's acetal linker. The libraries were rapidly synthesized in a parallel format and the generated compounds were tested as inhibitors of type 3 17β-HSD. Potent inhibitors were identified from these model libraries, especially six members of the level 3 library having at least one phenyl group. One of them, the 3β-(N-heptanoyl- -phenylalanine- -leucine-aminomethyl)-3α-hydroxy-5α-androstan-17-one (42) inhibited the enzyme with an IC₅₀ value of 227 nM, which is twice as potent as the natural substrate Δ⁴-dione when used itself as an inhibitor. Using the proliferation of androgen-sensitive (AR⁺) Shionogi cells as model of androgenicity, the compound 42 induced only a slight proliferation at 1 μM (less than previously reported type 3 17β-HSD inhibitors) and, interestingly, no proliferation at 0.1 μM.