Targeted migration of pherophorin‐S indicates extensive extracellular matrix dynamics in Volvox carteri

Hydroxyproline‐rich glycoproteins (HRGPs) constitute a major group of proteins of the extracellular matrix (ECM). The multicellular green alga Volvox carteri is a suitable model organism in which to study the evolutionary transition to multicellularity, including the basic principles and characteristics of an ECM. In Volvox, the ECM is dominated by a single HRGP family: the pherophorins. Our inventory amounts to 117 pherophorin‐related genes in V. carteri. We focused on a pherophorin with an unexpected characteristic: pherophorin‐S is a soluble, non‐cross‐linked ECM protein. Using transformants expressing a YFP‐tagged pherophorin‐S we observed the synthesis and secretion of pherophorin‐S by somatic cells in vivo, and we then traced the protein during its conspicuous migration to the ECM around prehatching juveniles and its localized concentration there. Our results provide insights into how an ECM zone surrounding the progeny is remotely affected by distantly located parental somatic cells. In view of the properties and migration of pherophorin‐S, we conclude that pherophorin‐S is likely to act as an ECM plasticizer to allow for dynamic ECM remodeling.     

Self-assembly and cross-linking of Volvox extracellular matrix glycoproteins are specifically inhibited by Ellman's reagent.

A major impediment to the biochemical characterization of extracellular matrices from algae (as well as higher plants) is the extensive covalent cross-linking that exists in the matrix, rendering most components insoluble and resistant to conventional extraction procedures. In the multicellular green alga Volvox, biogenesis of the extracellular matrix (ECM) is initiated immediately after the process of embryonic inversion. At this stage of development, the sulfhydryl reagent 5, 5'-dithio-bis(2-nitrobenzoic acid), known as Ellman's reagent, interferes in a highly specific manner with ECM biogenesis. Treated post-inversion embryos are no longer able to assemble an intact ECM and consequently dissociate into a suspension of single cells. Dissociated cells remain viable and continue to secrete ECM proteins into the growth medium, as documented by the identification of several members of the pherophorin family. Cross-linked ECM polymers such as sulfated surface glycoprotein 185 remain in a soluble state. Thus, treatment with Ellman's reagent opens a simple approach for the isolation and characterization of otherwise inaccessible monomeric precursors.     

Sex as a response to oxidative stress: stress genes co-opted for sex

Despite a great deal of interest, the evolutionary origins and roles of sex remain unclear. Recently, we showed that in the multicellular green alga, Volvox carteri, sex is a response to increased levels of reactive oxygen species (ROS), which could be indicative of the ancestral role of sex as an adaptive response to stress-induced ROS. To provide additional support for the suggestion that sex evolved as a response to oxidative stress, this study addresses the hypothesis that genes involved in sexual induction are evolutionarily related to genes associated with various stress responses. In particular, this study investigates the evolutionary history of genes specific to the sexual induction process in V. carteri--including those encoding the sexual inducer (SI) and several SI-induced extracellular matrix (ECM) proteins. Surprisingly, (i) a highly diversified multigene family with similarity to the V. carteri SI and SI-induced pherophorin family is present in its unicellular relative, Chlamydomonas reinhardtii (which lacks both a SI and an ECM) and (ii) at least half of the 12 identified gene members are induced (as inferred from reported expressed sequence tags) under various stress conditions. These findings suggest an evolutionary connection between sex and stress at the gene level, via duplication and/or co-option.     

Pherophorins: a family of extracellular matrix glycoproteins fromVolvox structurally related to the sex-inducing pheromone

Pherophorins are extracellular matrix (ECM) glycoproteins from Volvox that share homology with the sex-inducing pheromone. A novel pherophorin (pherophorin III) was characterized both with respect to expression pattern and proteolytic processing in vivo. Furthermore, it was shown that the pherophorins represent a protein family of ECM glycoproteins exhibiting a modular composition: their N-terminally located domain is a homolog of a domain found in the ECM glycoprotein SSG 185. Together with SSG 185, pherophorin I is a main component of the cellular zone within the ECM. The Volvox genome contains a tandem arrangement of genes encoding pherophorin II-related polypeptides. Inhibition of proteolytic processing of pherophorin II and III in vivo appears to result in the suppression of sexual induction.Abbreviations ECM extracellular matrix - PCR polymerase chain reaction - RACE rapid amplification of cDNA ends We wish to thank Dr. R. Deutzmann (Universität Regensburg, Germany) for sequencing peptides. This work was supported by the Deutsche Forschungsgemeinschaft (SFB43).     

Differential targeting of closely related ECM glycoproteins: the pherophorin family from Volvox.

The alga Volvox carteri represents one of the simplest multicellular organisms. Its extracellular matrix (ECM) is modified under developmental control, e.g. under the influence of the sex-inducing pheromone that triggers development of males and females at a concentration below 10(-16) M. A novel ECM glycoprotein (pherophorin-S) synthesized in response to this pheromone was identified and characterized. Although being a typical member of the pherophorins, which are identified by a C-terminal domain with sequence homology to the sex-inducing pheromone, pherophorin-S exhibits a completely novel set of properties. In contrast to the other members of the family, which are found as part of the insoluble ECM structures of the cellular zone, pherophorin-S is targeted to the cell-free interior of the spherical organism and remains in a soluble state. A main structural difference is the presence of a polyhydroxyproline spacer in pherophorin-S that is linked to a saccharide containing a phosphodiester bridge between two arabinose residues. Sequence comparisons indicate that the self-assembling proteins that create the main parts of the complex Volvox ECM have evolved from a common ancestral gene.     

Transcriptional activation by the sexual pheromone and wounding: a new gene family from Volvox encoding modular proteins with (hydroxy)proline-rich and metalloproteinase homology domains

The green alga Volvox represents the simplest kind of multicellular organism: it is composed of only two cell types, somatic and reproductive, making it suitable as a model system. The sexual development of males and females of Volvox carteri is triggered by a sex-inducing pheromone at a concentration of < 10-16 M. Early biochemical responses to the pheromone involve structural modifications within the extracellular matrix (ECM). By differential screenings of cDNA libraries made from mRNAs of pheromone-treated Volvox, four novel genes were identified that encode four closely related Volvox metalloproteinases that we use to define a new protein family, the VMPs. The existence of several features common to matrix glycoproteins, such as signal peptides, a (hydroxy)proline content of 12-25%, and Ser(Pro)2-4 repeats, suggest an extracellular localization of the VMPs within the ECM. Synthesis of VMP cDNAs is triggered not only by the sex-inducing pheromone, but also by wounding, and is restricted to the somatic cell type. Sequence comparisons suggest that the VMPs are members of the MB clan of zinc-dependent matrix metalloproteinases, although the putative zinc binding site of all VMPs is QEXXHXXGXXH rather than HEXXHXXGXXH. The presence of glutamine instead of histidine in the zinc binding motif suggests a novel family, or even clan, of peptidases. Like the matrixin family of human collagenases, Volvox VMPs exhibit a modular structure: they possess a metalloproteinase homology domain and a (hydroxy)proline-rich domain, and one of them, VMP4, also has two additional domains. Metalloproteinases seem to be crucial for biochemical modifications of the ECM during development or after wounding in the lower eukaryote Volvox with only two cell types, just as in higher organisms.     

Sexual pheromone induces diffusion of the pheromone-homologous polypeptide in the extracellular matrix of Volvox carteri

The C-terminal domain of pherophorin II is homologous to the sexual pheromone of Volvox carteri and is released from other domains during sexual induction. Green fluorescent protein fused to the C terminus of pherophorin II was located at the extracellular matrix directly surrounding the gonidium, the final target of the sexual-induction signal.     

The pherophorins: common, versatile building blocks in the evolution of extracellular matrix architecture in Volvocales

Green algae of the order Volvocales provide an unrivalled opportunity for exploring the transition from unicellularity to multicellularity. They range from unicells, like Chlamydomonas, through homocytic colonial forms with increasing cooperation of individual cells, like Gonium or Pandorina, to heterocytic multicellular forms with different cell types and a complete division of labour, like Volvox. A fundamental requirement for the evolution of multicellularity is the development of a complex, multifunctional extracellular matrix (ECM). The ECM has many functions, which can change under developmental control or as a result of environmental factors. Here molecular data from 15 novel proteins are presented. These proteins have been identified in Chlamydomonas reinhardtii, Gonium pectorale, Pandorina morum and Volvox carteri, and all belong to a single protein family, the pherophorins. Pherophorin-V1 is shown to be a glycoprotein localized to the 'cellular zone' of the V. carteri ECM. Pherophorin-V1 and -V2 mRNAs are strongly induced not only by the sex inducer, which triggers sexual development at extremely low concentrations, but also by mechanical wounding. Like the extensins of higher plants, which are also developmentally controlled or sometimes inducible by wounding, the pherophorins contain a (hydroxy-)proline-rich (HR) rod-like domain and are abundant within the extracellular compartment. In contrast to most extensins, pherophorins have additional globular A and B domains on both ends of the HR domains. Therefore pherophorins most closely resemble a particular class of higher plant extensin, the solanaceous lectins (e.g. potato lectin), suggesting multivalent carbohydrate-binding functions are present within the A and B domains and are responsible for cross-linking. Our results suggest that pherophorins are used as the building blocks for the extracellular scaffold throughout the Volvocales, with the characteristic mesh sizes in different ECM structures being a result of the highly diverse extensions of the HR domains. Pherophorins have therefore been a versatile element during the evolution of ECM architecture in these green algae.     

An extracellular matrix-localized metalloproteinase with an exceptional QEXXH metal binding site prefers copper for catalytic activity

The extracellular matrix (ECM) of the simple multicellular organism Volvox contains many region-specific morphological elements and mediates a variety of developmental and physiological responses by modification of its components. The fact that >95% of the mature organism is ECM makes Volvox suitable as a model system for ECM investigations. VMPs are a family of Volvox genes that are homologous to zinc-dependent matrix metalloproteinases (MMPs). Here we describe the identification and purification of the first VMP protein, VMP3. The 470-kDa VMP3 glycoprotein is localized within the ECM, and its biosynthesis is induced by the sex pheromone. The metal binding motif of VMP3 is QEXXH, not HEXXH as known for approximately 1300 other metalloproteinases. VMP3 shows proteinase activity and is inhibited by EDTA or the MMP inhibitor GM 6001, but in contrast to all known proteinases, VMP3 clearly prefers copper for activity rather than zinc. The exchange from Q to H within the QEXXH motif abolishes its copper preference. The unique properties of VMP3 suggest a novel type of metalloproteinase.     

Response to the sexual pheromone and wounding in the green alga volvox: induction of an extracellular glycoprotein consisting almost exclusively of hydroxyproline

The extracellular matrix (ECM) of Volvox is modified during development or in response to external stimuli, like the sex-inducing pheromone. It has recently been demonstrated that a number of genes triggered by the sex-inducing pheromone are also inducible by wounding. By differential screening of a cDNA library, a novel gene was identified that is transcribed in response to the pheromone. Its gene product was characterized as an ECM glycoprotein with a striking feature: it exhibits a hydroxyproline content of 68% and therefore is an extreme member of the family of hydroxyproline-rich glycoproteins (HRGPs). HRGPs are known as constituents of higher plant ECMs and seem to function as structural barriers in defense responses. The Volvox HRGP is also found to be inducible by wounding. This indicates that the wound response scenarios of higher plants and multicellular green algae may be evolutionary related.     

Comparative analysis of human matrix metalloproteinases: Emerging therapeutic targets in diseases

The identification of specific target proteins for any diseased condition involves extensive characterization of the potentially involved proteins. Members of a protein family demonstrating comparable features may show certain unusual features when implicated in a pathological condition. Advancements in the field of computational biology and the use of various bioinformatics tools for analysis can aid researchers to comprehend their system of work in primary stages of research. This initial screening can help to reduce time and cost of testing and experimentation in laboratory. Human matrix metalloproteinase (MMP) family of endopeptidases is one such family of 23 members responsible for the remodeling of extracellular matrix (ECM) by degradation of the ECM proteins. Though their role has been implicated in various pathological conditions such as arthritis, atherosclerosis, cancer, liver fibrosis, cardio-vascular and neurodegenerative disorders, little is known about the specific involvement of members of the large MMP family in diseases. A comparative in silico characterization of the MMP protein family has been carried out to analyze their physico-chemical, secondary structural and functional properties. Based on the observed patterns of occurrence of atypical features, we hypothesize that cysteine rich and highly thermostable MMPs might be key players in diseased conditions. Thus, a plausible grouping of disease responsive MMPs that might be considered as promising clinical targets may be done. This study can be used as a fundamental approach to characterize, analyze and screen large protein families for the identification of signature patterns.     

ADAMTS: a novel family of extracellular matrix proteases

ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) is a novel family of extracellular proteases found in both mammals and invertebrates. Members of the family may be distinguished from the ADAM (a disintegrin and metalloprotease) family members based on the multiple copies of thrombospondin 1-like repeats they carry. With at least nine members in mammals alone, the ADAMTS family members are predicted by their structural domains to be extracellular matrix (ECM) proteins with a wide range of activities and functions distinct from members of the ADAM family that are largely anchored on the cell surface. ADAMTS2 is a procollagen N-proteinase, and the mutations of its gene are responsible for Human Ehlers-Danlos syndrome type VII C and bovine dermatosparaxis. ADAMTS4 and ADAMTS5 are aggrecanases implicated in the degradation of cartilage aggrecan in arthritic diseases. Other members of the ADAMTS family have also been implicated in roles during embryonic development and angiogenesis. Current and future studies on this emerging group of ECM proteases may provide important insights into developmental or pathological processes involving ECM remodeling.     

Small leucine rich proteoglycan family regulates multiple signalling pathways in neural development and maintenance

The small leucine-rich repeat proteoglycan (SLRPs) family of proteins currently consists of five classes, based on their structural composition and chromosomal location. As biologically active components of the extracellular matrix (ECM), SLRPs were known to bind to various collagens, having a role in regulating fibril assembly, organization and degradation. More recently, as a function of their diverse proteins cores and glycosaminoglycan side chains, SLRPs have been shown to be able to bind various cell surface receptors, growth factors, cytokines and other ECM components resulting in the ability to influence various cellular functions. Their involvement in several signaling pathways such as Wnt, transforming growth factor-β and epidermal growth factor receptor also highlights their role as matricellular proteins. SLRP family members are expressed during neural development and in adult neural tissues, including ocular tissues. This review focuses on describing SLRP family members involvement in neural development with a brief summary of their role in non-neural ocular tissues and in response to neural injury.     

Adhesion-modulating/matricellular ECM protein families: a structural, functional and evolutionary appraisal

The thrombospondins are a family of secreted, oligomeric glycoproteins that interact with cell surfaces, multiple components of the extracellular matrix, growth factors and proteases. These interactions underlie complex roles in cell interactions and tissue homeostasis in animals. Thrombospondins have been grouped functionally with SPARCs, tenascins and CCN proteins as adhesion-modulating or matricellular components of the extracellular milieu. Although all these multi-domain proteins share various commonalities of domains, the grouping is not based on structural homologies. Instead, the terms emphasise the general observations that these proteins do not form large-scale ECM structures, yet act at cell surfaces and function in coordination with the structural ECM and associated extracellular proteins. The designation of adhesion-modulation thus depends on observed tissue and cell culture ECM distributions and on experimentally identified functional properties. To date, the evolutionary relationships of these proteins have not been critically compared: yet, knowledge of their evolutionary histories is clearly relevant to any consideration of functional similarities. In this article, we survey briefly the structural and functional knowledge of these protein families, consider the evolution of each family, and outline a perspective on their functional roles.     

VCRPs,small cysteine-rich proteins,might be involved in extracellular signaling in the green alga Volvox

The sex-inducer of the spherical green alga Volvox carteri is one of the most potent biological effector molecules known: it is released into the medium by sexual males and triggers the switch to the sexual cleavage program in the reproductive cells of vegetatively grown males and females even at concentrations as low as 10^-16 M. In an adult Volvox alga, all cells are embedded in an extensive extracellular matrix (ECM), which constitutes >99% of the volume of the spheroid. There exist no cytoplasmic connections between the cells in an adult alga, so any signal transduction between different cells or from the organism''s environment to a reproductive cell must involve the ECM. Recently, a small cysteine-rich extracellular protein, VCRP, was identified in Volvox and shown to be quickly synthesized by somatic cells in response to the sex-inducer. Due to its characteristics, VCRP was speculated to be an extracellular second messenger from somatic cells to reproductive cells. Here a related protein, VCRP2, is presented, exhibiting a 56% amino acid sequence identity with VCRP. Two possible scenarios for signal transduction from the sex-inducer to the reproductive cell are discussed.Key words: cell wall, extracellular matrix, extracellular second messenger, green algae, sex-inducer, sex inducing pheromone, sexual development, stress response, Volvocaceae, wounding     

Evolution of prokaryotic SPFH proteins

Background  

The SPFH protein superfamily is a diverse family of proteins whose eukaryotic members are involved in the scaffolding of detergent-resistant microdomains. Recently the origin of the SPFH proteins has been questioned. Instead, convergent evolution has been proposed. However, an independent, convergent evolution of three large prokaryotic and three eukaryotic families is highly unlikely, especially when other mechanisms such as lateral gene transfer which could also explain their distribution pattern have not yet been considered.     

Matrix metalloproteinases and epidermal wound repair

Epidermal wound healing is a complex and highly coordinated process where several different cell types and molecules, such as growth factors and extracellular matrix (ECM) components, play an important role. Among the many proteins that are essential for the restoration of tissue integrity is the metalloproteinase (MMP) family. MMPs can act on ECM and non-ECM components affecting degradation and modulation of the ECM, growth-factor activation and cell–cell and cell–matrix signalling. MMPs are secreted by different cell types such as keratinocytes, fibroblasts and inflammatory cells at different stages and locations during wound healing, thereby regulating this process in a very coordinated and controlled way. In this article, we review the role of MMPs and their inhibitors (TIMPs), as well as the disintegrin and metalloproteinase with the thrombospondin motifs (ADAMs) family, in epithelial wound repair.     

The role of motor proteins in endosomal sorting

Microtubule motor proteins play key roles in the spatial organization of intracellular organelles as well as the transfer of material between them. This is well illustrated both by the vectorial transfer of biosynthetic cargo from the endoplasmic reticulum to the Golgi apparatus as well as the sorting of secretory and endocytic cargo in the endosomal system. Roles have been described for dynein and kinesin motors in each of these steps. Cytoplasmic dynein is a highly complex motor comprising multiple subunits that provide functional specialization. The family of human kinesins includes over 40 members. This complexity provides immense functional diversity, yet little is known of the specific requirements and functions of individual motors during discrete membrane trafficking steps. In the present paper, we describe some of the latest findings in this area that seek to define the mechanisms of recruitment and control of activity of microtubule motors in spatial organization and cargo trafficking through the endosomal network.     

The role of matricellular proteins in glaucoma

Glaucoma is an optic neuropathy affecting approximately 60 million people worldwide and is the second most common cause of irreversible blindness. Elevated intraocular pressure (IOP) is the main risk factor for developing glaucoma and is caused by impaired aqueous humor drainage through the trabecular meshwork (TM) and Schlemm's canal (SC). In primary open angle glaucoma (POAG), this elevation in IOP in turn leads to deformation at the optic nerve head (ONH) specifically at the lamina cribrosa (LC) region where there is also a deposition of extracellular matrix (ECM) molecules such as collagen and fibronectin.Matricellular proteins are non-structural secreted glycoproteins that help cells communicate with their surrounding ECM. This family of proteins includes connective tissue growth factor (CTGF), also known as CCN2, thrombospondins (TSPs), secreted protein acidic and rich in cysteine (SPARC), periostin, osteonectin, and Tenascin-C and -X and other ECM proteins. All members appear to play a role in fibrosis and increased ECM deposition. Most are widely expressed in tissues particularly in the TM and ONH and deficiency of TSP1 and SPARC have been shown to lower IOP in mouse models of glaucoma through enhanced outflow facility. The role of these proteins in glaucoma is emerging as some have an association with the pathophysiology of the TM and LC regions and might therefore be potential targets for therapeutic intervention in glaucoma.     

Lefty contributes to the remodeling of extracellular matrix by inhibition of connective tissue growth factor and collagen mRNA expression and increased proteolytic activity in a fibrosarcoma model

Homeostasis of the extracellular matrix (ECM) of tissues is regulated by controlling deposition and degradation of ECM proteins. The breakdown of ECM is essential in blastocyst implantation and embryonic development, tissue morphogenesis, menstrual shedding, bone formation, tissue resorption after delivery, and tumor growth and invasion. TGF-beta family members are one of the classes of proteins that actively participate in the homeostasis of ECM. Here, we report on the effect of lefty, a novel member of the TGF-beta family, on the homeostasis of extracellular matrix in a fibrosarcoma model. Fibroblastic cells forced to express lefty by retroviral transduction lost their ability to deposit collagen in vivo. This event was associated with down-regulation of the steady-state level of connective tissue growth factor that induces collagen type I mRNA. In addition, lefty transduction significantly decreased collagen type I mRNA expression and simultaneously increased collagenolytic, gelatinolytic, elastolytic, and caseinolytic activities in vivo by the transduced fibroblasts. These findings provide a new insight on the actions of lefty and suggest that this cytokine plays an active role in remodeling of the extracellular matrix in vivo.