Glycobiologyvol.17 no.9pp.897–905,2007 doi:10.1093/glycob/cwm043 Advance Access publication on April 18, 2007 Perlecan—a multifunctional extracellular scaffold

Mary C Farach-Carson and Daniel D Carson1 well as pathologies. The normal processes of embryo implan- Department of Biological Sciences, University of Delaware, Newark, tation and placentation are quite complex and require the full DE 19716 spectrum of these activities. Therefore, this system provides an excellent platform to examine perlecan functions. This Downloaded from https://academic.oup.com/glycob/article/17/9/897/1987692 by guest on 29 September 2021 Received on February 8, 2007; revised on April 5, 2007; accepted on April 5, 2007 review will discuss the key events of embryo implantation and placentation, the complexities of perlecan biology, and Perlecan is a large multidomain proteogly- the potential functions of perlecan as an extracellular scaffold can of the . Expression of this proteo- modulating events occurring in early mammalian preg- glycan changes dynamically during embryo implantation nancy and development. and placentation. Perlecan is expressed by various cells of the embryo including trophectoderm and trophoblast as Embryo implantation and placentation well as the maternal compartment, including basal lamina underlying uterine epithelia and endothelia and, Embryo implantation is a well-coordinated process in which most dynamically, in developing decidua. Perlecan sup- the uterus matures to a state in which it can support embryo ports various biological functions, including , attachment. This maturation is controlled by the actions of growth factor binding, and modulation of apoptosis. ovarian steroids produced by corpora lutea. This endocrine Moreover, studies in other systems demonstrate that perle- mechanism links preimplantation embryonic development to can expression and activity can be controlled at many uterine development. The mammalian egg is released from levels, including transcription, alternative splicing, and the mature follicle, leaving behind the hormone-producing extracellular proteolysis. This review will discuss changes corpus luteum. Following fertilization, the single-cell zygote in perlecan expression that occur during embryo implan- undergoes a series of cell divisions within the tation and placentation. Furthermore, we propose a coat of the zona pellucida, ultimately giving rise to the blasto- model in which perlecan represents an extracellular scaf- cyst, a fluid-filled structure containing the cells of the inner cell fold protein that supports complex, distinct functions in mass and surrounded by a layer of trophectoderm. After hatch- its full-length form or smaller forms generated by alter- ing from the zona pellucida, the trophectoderm will mediate native mRNA splicing, extracellular proteolysis, or glycosi- embryo attachment to and invasion of the endometrium, and dase action. subsequently develop into hormone-producing trophoblast and form the placenta. The placenta itself is a complex Keywords: perlecan/heparan sulfate/embryo implantation/ organ consisting of several trophoblastic cell types, which pro- placentation vides hormonal signals and support fetal nutrition and waste exchange by establishing contact with the maternal blood supply. Reviews of these processes, including differences Introduction that occur among species, are available (Carson et al. 2000; Chaddha et al. 2004; Enders and Carter 2006). Heparan sulfate (HSPGs) are defined as The endometrium undergoes a cyclic process of differen- containing one or more covalently attached heparan sulfate tiation in response to the actions of ovarian steroids. This (HS) chains. HSPGs can be divided into three major classes: involves proliferation of both epithelial and stromal elements (1) lipid-anchored, e.g. the ; (2) transmembrane, leading to endometrial growth. Toward the latter half of this e.g. the syndecans; and (3) extracellular or secreted. The cyclical process, the endometrium becomes transiently recep- latter class includes several unrelated proteoglycan cores, tive with regard to embryo attachment, after which point it including type XVIII, agrin, and perlecan. Perlecan transitions to a refractory state wherein implantation cannot is a large multifunctional HSPG found in virtually all basal occur until hormone levels fall and the endometrial cycle lamina as well as in the interstitial matrix of certain tissues, can again start. Thus, the attachment-competent blastocyst including cartilage (Farach-Carson et al. 2005), bone stroma must arrive in the uterus during this “window” of uterine (Schofield et al. 1999), and uterine decidua (French et al. receptivity. Minimally, the receptive uterine state requires 1999). In addition to its structural role, both the HS constitu- that the epithelial cells lining the luminal surface be capable ents and the protein core of perlecan support multiple biologi- of binding the attachment-competent blastocyst. A variety of cal activities relevant to many processes that occur during adhesion-promoting molecules have been described at the sur- normal tissue homeostasis and embryonic development as faces of attachment-competent blastocysts and receptive phase uterine epithelia, including HSPGs and their binding proteins 1To whom correspondence should be addressed; Tel: þ1-302-831-4296; Fax: [reviewed in Carson et al. (2000)]. Nonetheless, knockout þ1-302-831-2281; e-mail: [email protected] studies in mice indicate that none of these candidates are

# TheAuthor2007.PublishedbyOxfordUniversityPress.Allrightsreserved.Forpermissions,pleasee-mail:[email protected] 897 MC Farach-Carson and DD Carson absolutely required to support the initial events of embryo data indicates that perlecan protein expression is robust and attachment. This indicates that there is considerable redun- persistent. Together, these observations suggest that perlecan dancy in the systems involved in early embryo implantation. protein is quite stable in this environment, in spite of the exten- The attachment systems which embryos can utilize in various sive metalloprotease-dependent remodeling that occurs species have been reviewed (Carson et al. 2000; Aplin and (Murray and Lessey 1999; Schatz et al. 1999). As indicated Kimber 2004). In response to embryo attachment, the uterine in Figure 2, perlecan is cleaved at discrete sites by certain stroma undergoes a differentiation process resulting in the for- matrix metalloproteases (MMP), including MMP3 (stromely- mation of decidua. Decidual tissue formation includes both sin), MMP1 (collagenase), and plasmin (Whitelock et al. extracellular matrix (ECM) remodeling and de novo expression 1996), as well as by the cell surface proteases, membrane- of ECM components, not usually found in interstitial ECM, type MMPs (d’Ortho et al. 1997). More recently, the C- including , collagen type IV, and perlecan [reviewed terminal portion of perlecan has been shown to be a substrate in Murray and Lessey (1999) and Schatz et al. (1999)]. for bone morphogenetic protein (BMP)-1/Tolloid-like metal- loproteases (Gonzalez et al. 2005). It is not clear if the perle- Downloaded from https://academic.oup.com/glycob/article/17/9/897/1987692 by guest on 29 September 2021 can present in endometrial tissues is primarily in an intact Perlecan expression in embryo implantation form, is cleaved or if cleavage only occurs in select regions and placentation of decidua. Experiments should be performed to test these pos- Most of the available data on perlecan expression during early sibilities, especially because certain perlecan fragments may mammalian embryonic development have been derived from have activities distinct from those of intact protein. For studies of mice. Perlecan is first detected at the four- to example, a C-terminal fragment released from endothelial eight-cell stage (Dziadek et al. 1985); however, this expression cell-derived perlecan protects fibroblasts from apoptosis is transient because other studies indicate that perlecan is not (Laplante et al. 2006) and may play a similar role in expressed in blastocysts until after hatching from the zonae decidua. A similar fragment (endorepellin) has been shown pellucidae (Smith et al. 1997). This timing parallels increases to be antiangiogenic (Gonzalez et al. 2005) and could contrib- in HSPG synthesis (Farach et al. 1987). At this point, perlecan ute to the lack of vasculature observed in primary decidual is found on the external surface of the trophectoderm, i.e. at a tissue (Parr et al. 1986). position where it could participate in blastocyst attachment. In The high level of pericellular perlecan expression in decidua this regard, HS-dependent interactions support embryo attach- may impact implantation and placentation at several levels. ment to various substrates, including laminin, fibronectin, and Through its ability to bind growth factors and cytokines, per- primary cultures of uterine epithelial cells (Farach et al. 1987). lecan can control delivery of these proteins by limiting their It is not clear which cell surface components retain perlecan at diffusion from local sites of expression and concentrating the trophectodermal cell surface; however, b1- and b3- them at the site of rapid embryo development, trophoblast containing integrins have been identified as perlecan receptors differentiation, and placental formation. Perlecan also is in other systems (Hayashi et al. 1992; Brown et al. 1997) and likely to play an important role in neo-vascularization associ- these integrin complexes have been identified on the trophec- ated with embryo implantation and placental development. toderm surface (Armant 2005). As mentioned above, the initial Perlecan is well known to bind and enhance the activities of interactions with the uterine epithelial surface appear to be HS angiogenic growth factors such as vascular endothelial dependent. In this regard, the transmembrane form of heparin growth factor (VEGF) and fibroblast growth factors (FGFs) binding-epidermal growth factor (HB-EGF) is induced locally (Jiang and Couchman 2003) and, thus, is likely to promote at embryo implantation sites (Das et al. 1994). Other candidate the massive tissue vascularization that occurs during placenta- HS-bindingmoleculesexpressedbyuterineepithelialcellsur- tion. Moreover, endothelial cells directly bind to the perlecan faces include amphiregulin (Das et al. 1995) and HIP/RPL29 protein core (Hayashi et al. 1992). This latter activity is modu- (Rohde et al. 1996). Null mutants have been generated to all of lated by the presence of chains. Thus, these proteins and none display an overt implantation defect, ECM remodeling enzymes, heparanases or metalloproteases, again probably due to redundancy in function (Luetteke that also are abundantly expressed in decidua can convert per- et al. 1999; Iwamoto et al. 2003; Kirn-Safran, Oristian, et al. lecan from a form that may stimulate endothelial 2007; Kirn-Safran, Oristian, Focht, et al. 2007). It should be and migration to forms that better support stable endothelial noted that all of these proteins may participate in implantation. cell adhesion and inhibit further endothelial cell migration Loss of any one (or two) of these proteins may still leave and proliferation, e.g. by the production of endorepellin enough HS-binding activity to support this process due to (Gonzalez et al. 2005). redundancy of function. It also is possible that expression of Perlecan expression has been examined in developing other HS-binding proteins is elevated when one is lost. human placentae (Rohde et al. 1998; Yang et al. 2005). As Finally, HS-binding proteins that have not yet been identified expected, it is found in all basal laminae of these structures; or studied in the context of implantation may play key roles. however, perlecan also accumulates in Nitabuch’s membrane Thus, considerably more work will need to be done to assess (Rohde et al. 1998), an area of attachment at the maternal– fully the role of HS-dependent interactions during early fetal interface placental villi and the decidua (Figure 1). stages of embryo implantation. Curiously, perlecan expression is elevated and the glycosami- Perlecan is induced locally in decidual tissue surrounding noglycan composition is altered in placentae in women with the implantation site, first in the primary decidua and later in mellitus (Chen et al. 2007). In the latter case, there thesecondarydecidualzone(Figure1;Frenchetal.1999). is an increase in the chondroitin/dermatan sulfate content in Insituhybridizationresultsindicatethatincreasesinperlecan the diabetic placentae. The functional significance of these mRNA expression are transient; however, immunostaining observations is not clear, but it has been suggested that these 898 Perlecan—a multifunctional extracellular proteoglycan scaffold Downloaded from https://academic.oup.com/glycob/article/17/9/897/1987692 by guest on 29 September 2021

Fig. 1. Perlecan expression in implantation sites, embryos, and the fetal–maternal interface. The left hand panel shows the distribution of perlecan by immunostaining in a day 8-mouse implantation site (green) as nuclei are counter-stained with Draq 5 (blue). Perlecan is evident in basal lamina throughout the secondary decidual zone (Sdz), myometrium (Myo), ectoplacental cone (Epc), and developing lacunae (Lac). Perlecan also is evident at the interface of embryonic trophoblast and maternal tissue (white arrows) and Reichert’s membrane within the embryo (green arrow) as well as in the embryo and associated trophoblast. The right two panels were modified from Rohde and collaborators (1998) and show a low (A) and higher (B) magnification image of an 18-week placental villus (av) attached to the maternal tissue (s) stained with antibodies to perlecan (red) and HIP/RPL29 (green). In panel (A), note the intense staining for perlecan at the site of attachment (filled arrow) and underlying maternal tissue representing Nitabuch’s membrane (open arrow). Panel (B) is an area shown by the boxed area in panel (A) and demonstrates strong perlecan staining at villar attachment sites even where this is less obvious at lower magnification (reproduced from Rohde et al. (1998) with permission).

changes may be associated with changes in placental structure. endometrial vasculature is (Akerlund 1994; Sastry 1997). Another report, based entirely on immunostaining of mouse Thus, proper uterine innervation would be required not only embryos, indicates that perlecan expression is decreased by for myometrial contractile activity, but also for controlling interferon-g (Fontana et al. 2004). In spite of the nonquantita- uterine blood flood in support of the developing embryo and tive nature of these studies, they nonetheless are consistent fetus. Defects in controlling proper uterine blood flow underlie with observations in cell lines indicating that interferon-g preeclampsia, although no clear relationship between pree- potently inhibits perlecan promoter activity (Sharma and clampsia and perlecan expression, function, or processing Iozzo 1998). A prediction of these studies is that inflammation has been determined. that can occur in response to infection during pregnancy would Other studies indicate that Drosophila perlecan defects can reduce perlecan expression and contribute to spontaneous be rescued by exogenous FGF-2 and Indian hedgehog; abortion and recurrent miscarriage associated with this state however, binding studies indicate that hedgehog binds to per- (Raghupathy 2001; Laird et al. 2006). Relatively, little is lecan in a HS-independent manner, presumably through inter- known about transcriptional regulation of perlecan. Such actions with the protein core (Park et al. 2003). Hedgehog studies are needed in the context of endometrial and placental proteins also bind to perlecan and initiate signals in mamma- biology since they might identify pathways and potential thera- lian cells (Datta et al. 2006). Collectively, these observations peutic approaches to maintain perlecan expression during indicate that perlecan plays a role in both HS-dependent and inflammatory challenge. -independent signaling by controlling gradients of secreted factors regulating cell proliferation. From another perspective, Indian hedgehog is expressed in uterine epithelium and is a Physiological consequences of perlecan mutations progesterone-responsive gene (Takamoto et al. 2002). While Perlecan mutations have been created or identified in a number Indian hedgehog expression appears to decrease shortly after of species ranging from fruit flies to humans. Although embryo attachment, a series of other HS-binding proteins, par- implantation and placentation does not occur in the nonmam- ticularly those of the BMP/BMP-antagonist family are malian species, the defects observed in these cases may be induced robustly in decidua where perlecan probably plays a instructive when thinking about the potential impact on mam- role in restricting their diffusion (Paria et al. 2001). Thus, fea- malian reproductive events. In Drosophila, perlecan mutations tures of perlecan function determined in studies of lower occur in a gene called terribly reduced optic lobes or trol animals have parallels in uterine physiology. (Voigt et al. 2002). The trol mutations appear to be manifest In Caenorhabditis elegans, perlecan has been identified as primarily in the nervous system, acting downstream of an anti- the product of the UNC-52 gene (Rogalski et al. 2001). Lack proliferative gene, Ana, a secreted protein with antiprolifera- of perlecan/UNC-52 in nematodes results in severe body- tive activity. The trol phenotype results in failure of wall muscle defects and appears to be related to abnormal for- quiescent neuroblasts to initiate proliferation resulting in mation of integrin-containing complexes, although it is not small eyes and brains; however, this defect can be overcome clear if perlecan interactions in this regard are direct or indirect by increasing the expression of string, a Cdc25 ortholog [reviewed in Rogalski et al. (2001)]. Perlecan is highly (Park et al. 2003). While the placenta is not innervated, the expressed in basal lamina underlying uterine longitudinal 899 MC Farach-Carson and DD Carson Downloaded from https://academic.oup.com/glycob/article/17/9/897/1987692 by guest on 29 September 2021

Fig. 2. Perlecan as a scaffold—functional uncoupling by proteolysis. The figure diagrams perlecan and indicates domains I–V as well as major structural subdomains. The scissors indicate the positions of predicted sites of cleavage by the corresponding enzymes. Thrombin sites are indicated in grey. Although thrombin-sensitive sites are predicted by sequence inspection, these sites appear to be cryptic because intact perlecan is not sensitive to thrombin cleavage (Whitelock et al. 1996). and circular myometrial cells as well as uterine vasculature. likely. Recent studies indicate that perlecan variants occur in Therefore, perlecan is likely to participate in neuromuscular cartilage although it is not clear if this results from proteolysis, junction organization and control of uterine blood flow, as alternative splicing or both (Melrose et al. 2006). Moreover, mentioned above. In addition, UNC-52 defects impact the mammalian mutations in Smu-1 also result in increased developmental program of gonads apparently through expression of perlecan splice variants suggesting that this impaired growth factor signaling (Merz et al. 2003). UNC- process is conserved throughout evolution (Sugaya et al. 52 gene products can occur as one of three major size variants 2006). As mentioned above, given the different functions generated by alternative splicing due to the presence of poly- associated with different perlecan domains, a systematic exam- adenylation sites downstream of (Dziadek et al. 1985; ination of perlecan splice variants as well as proteolytic Merz et al. 2003; Yang et al. 2005). The shortest form (S) processing in fetal–placental tissues is warranted. includes domains I–III, the intermediate-size form (M) con- Mouse null mutations in perlecan have been created, but tains domains I–IV, while the longest form (L) contains all display massive developmental abnormalities affecting the five domains. Additional minor splice variants have been heart, brain, kidney, and skeletal tissues, among others detected that yield differences in domains III and IV as well (Arikawa-Hirasawa et al. 1999; Costell et al. 1999). Most as a potential for .50 splice variants [reviewed in Mullen nulls die around gestational day 11.5, although a small percen- et al. (1999); and Rogalski et al. (2001)]. In this system, tage of pups go to term and die shortly afterwards. several RNA-binding proteins have been identified by Surprisingly, placental development appears normal in these genetic screens, which appear to regulate UNC-52 alternative animals, indicating that perlecan function is dispensible for splicing, including Smu-1, Smu-2, and Mec-8 (Spike et al. development of this tissue. The fact that the few nulls that 2002; Spartz et al. 2004). C. elegans mutants have been gen- go to term die shortly after birth makes it impossible to erated that prevent formation of the L and M isoforms and assess the role of perlecan in uterine physiology. demonstrate that domain V is not essential for normal Nonetheless, these observations demonstrate that blastocysts muscle formation and function; however, mutations that elimi- do not need to express perlecan to implant. This is similar to nate the M and L isoforms phenocopy UNC-52/perlecan nulls, observations made with many other null mutations of ECM demonstrating that the S-form cannot support the associated components or cell-adhesion molecules which collectively biological activity (Merz et al. 2003). Because the S-isoform indicate that there is considerable redundancy in embryo would carry as much glycosaminoglycan as the M isoform, attachment systems [reviewed in Carson et al. (2000)]. A these observations demonstrate that interactions with domain mouse perlecan mutant has been created that lacks the glyco- IV are essential. saminoglycan attachment sites in domain I (Rossi et al. 2003). Mammalian perlecan also is a large complex gene with 94 These animals have defects in lens and kidney development; exons (Cohen et al. 1993) and, therefore, has great potential however, no detailed information about placental or uterine to undergo alternative splicing; however, only one report of development or function has been reported. Careful studies alternative splicing of perlecan in mammalian systems has of the fertility, implantation success, and decidua formation appeared (Joseph et al. 1996). It is not clear how widespread in these mice is warranted. Nonetheless, because these alternative splicing is for perlecan, although this seems mutants have been successfully bred with collagen XVIII 900 Perlecan—a multifunctional extracellular proteoglycan scaffold null mice, it appears that they are at least partially fertile (Rossi a subset of the perlecan domains, and a relatively small et al. 2003). Two human mutations in perlecan are known, portion of the total molecule. The exact functions of the namely dyssegmental dysplasia, Silverman-Handmaker type other domains are not well understood, although they offer a [DDSH; MIM#224410; Arikawa-Hirasawa et al. (2001)] and wide variety of conserved structural motifs with broad func- Schwartz-Jampel syndrome type I [SJSI; MIM#255800; tional potential. A number of authors, including ourselves, Stum et al. (2006)]. Both are rare, autosomal recessive diseases have attempted to develop an integrated model that globally resulting in perlecan truncation mutants. DDSH newborns defines the function of perlecan in the various tissues where display massive skeletal abnormalities and die shortly after it plays a crucial role in essential processes such as organogen- birth. SJSI is represented by a spectrum of mutations with esis, wound healing, and (Farach-Carson et al. most patients reaching adulthood with a spectrum of disorders 2005; Iozzo 2005). Recently, Knox and collaborators (Knox related to the degree of haploinsufficiency, notably severe and Whitelock 2006) suggested that the context of the local muscle stiffness (Stum et al. 2005). No systematic studies on extracellular environment defines the function and downstream fertility of SJSI patients or the placentae for either DDSH or effects of perlecan on tissue structure and function. It also has Downloaded from https://academic.oup.com/glycob/article/17/9/897/1987692 by guest on 29 September 2021 SJSI are available. Recently, the mouse knockin model of been suggested that degradation of intact perlecan generates SJSI was created with a musculoskeletal phenotype similar bioactive fragments such as endorepellin with unique activities to that of humans; however, no information on the impact of distinct from those of the parent HS proteoglycan (Bix et al. this mutation on implantation or placentation is yet available 2004). Degradation of perlecan may also play a dynamic role (Rodgers et al. 2007). in reproductive processes. For example, selective destruction of perlecan occurs during ovulation in the focal intraepithelial matrix that develops between granulosa cells (focimatrix) and Perlecan activities the follicular basal lamina in ovarian follicles (Irving-Rodgers Perlecan contains three or more HS chains in unique domain I, et al. 2006). Amniotic fluid also has been shown to contain a and an additional glycosaminoglycan attachment site in C-terminal fragment of perlecan that may provide a useful domain V, approximately 4000 amino acids away (Figure 3). prognostic indicator for patients at risk of premature rupture Individual perlecan domains have unique abilities to interact of the fetal membrane (Thadikkaran et al. 2005). Dynamic with various heparin-binding (HB) growth factors such as changes in perlecan expression also occur in the region of VEGF, HB-EGF, and FGF-2; hedgehog, ECM, and basement the uterus near the implanting embryo in the peri-implantation membrane components; and the extracellular domains of cell period with loss of perlecan reported in the endometrial stroma surface components, together defining the complex perlecan (San Martin et al. 2004) and in the outer surface of the “interactome” (Figure 4). Some interactions are heparin/HS- embryonic trophectoderm after the initial stage of embryo dependent, some are heparin-influenced, and still others attachment (Carson et al. 1993). It is not clear whether the dra- involve core protein interactions that occur independently of matic reductions in perlecan in all of these cases occur in order heparin/HS. The function of perlecan as a co-receptor media- to generate active functional fragments of perlecan, to remove ting heparin-binding growth factor delivery and receptor sig- a function of the intact proteoglycan, or a combination of both naling has been well discussed (Jiang and Couchman 2003; effects that may accompany tissue remodeling. Fjeldstad and Kolset 2005), but these functions involve only

Perlecan—an extracellular scaffold A major unanswered question is why nature designed the large perlecan molecule as a composite of close to 50 connected, but independently folding, protein modules rather than as a series of smaller proteins. What advantage does linking these

Fig. 3. Perlecan structure. Composite scale model for intact human perlecan domains based on available images obtained using rotary shadowing of individually expressed domains and atomic force microscopy (see text). Unlike Fig. 4. Perlecan as a scaffold—domains and interactions. The top portion of linear models, the molecule appears as a modular structure with both globular the figure shows the five perlecan domains presented in Figure 3 separated into and extended regions that support its function as an extracellular scaffold individual segments. Below each domain is a list of proteins known to bind to protein. that region. For further details, see text. 901 MC Farach-Carson and DD Carson functional units confer? As discussed elsewhere in this article, glycosaminoglycan attachment site (Friedrich et al. 1999) and it is not clear to what extent mammalian tissues produce per- consists of laminin G and EGF-like domains. The laminin G lecan isoform variants. Conditions that favor increased tran- domain contains a “jellyroll fold” with hydrophobic core resi- scription and translation, or stabilize the functional lifetime dues located in central beta strands, whereas the EGF-like of perlecan typically will generate a very large secreted domain is formed from two-stranded b-sheets followed by a protein with the potential to support many complex activities. loop to a C-terminal short-two-stranded sheet; such domains While the large domain IV of perlecan has been suggested to usually contain three disulfides. Endorepellin has been reported function as a “complex cluster of heterotypic interaction sites” to potently inhibit four aspects of angiogenesis, including endo- supporting ECM assembly (Hopf et al. 2001), the concept of thelial cell migration, collagen-induced endothelial tube mor- the complete perlecan structure as an extracellular scaffolding phogenesis, and blood vessel growth in the chorioallantoic protein modulating signaling pathways in target cells has not membrane and in Matrigelw plug assays (Mongiat et al. been explored. This is true despite the provocative hint of 2003). The function of this domain in preventing angiogenic this function from two independent observations. First, invasion is an intriguing possibility. Downloaded from https://academic.oup.com/glycob/article/17/9/897/1987692 by guest on 29 September 2021 recent data indicate that perlecan contributes to the assembly Unlike previous linear depictions, Figure 3 shows a new of acetylcholine receptor clusters and influences development composite model for intact perlecan based on available and signaling within the neuromuscular junction (Smirnov images obtained using rotary shadowing of individually et al. 2005). Second, human mutations producing perlecan expressed domains and atomic force microscopy haploinsufficiency in the ECM manifest as SJSI, which is (Chakravarti et al. 1995; Costell et al. 1996; Brown et al. characterized by disruptions in cellular electrical activity 1997; Dolan et al. 1997; Hopf et al. 1999; Chen and (Cao et al. 1978). Targeted reduction of perlecan expression Hansma 2000). As shown, the predicted dimensions of the alters intracellular signaling (Savore et al. 2005), including intact perlecan molecule span a distance of some 100– signaling pathways known to originate in “signalosomes” 200 nm depending on the degree of twisting, especially of held within lipid rafts (Chu et al. 2004). Thus, we undertook the long-Ig repeat modules in domain IV that span some a structure and function analysis using published literature 60–80 nm. Domain II spans an average distance of 18 nm and public databases to explore the concept that perlecan has and domains III and V each span approximately 20 nm. the necessary properties to function as an extracellular scaffold Domain I is the smallest, but is made larger by the presence that might directly link the ECM to the cell surface, particu- of the extended glycosaminoglycan chains. To place these larly at sites at which cellular signals are initiated. dimensions into the context of perlecan as a putative extra- Figure 2 shows a representation of the domain structures of cellular scaffolding protein, a typical lipid raft signaling micro- perlecan in addition to the proteolytic sites. Domains and sub- domain has an average diameter of 6–20 nm. Of interest, the domains are color-coded and descriptions may be found in size of these rafts is thought to be able to increase to 100– Pfam (http://www.sanger.ac.uk/Software/Pfam/). Domain 200 nm by coalescence and stabilization of smaller rafts that I contains the three glycosaminoglycan attachment sites and are cross linked (Edidin 2003). The size of these rafts has the sea urchin sperm protein-enterokinase-agrin (SEA) module been proposed to influence complex signaling cascades that (Dolan et al. 1997). Domain II contains a low-density lipopro- are scaffolded in plasma membrane microdomains (Nicolau tein (LDL) receptor domain class A motif which is character- et al. 2006). Furthermore, functional receptors in myocytes ized by six disulfide-bound cysteines and a highly conserved have been shown to be organized into multiprotein domains cluster of negatively charged amino acids, of which many of approximately 140 nm average diameter (Ianoul et al. are clustered on one face of the module; this domain is 2005). Hence, it is very intriguing to speculate that a known to be both LDL and calcium binding (Costell et al. complex ECM molecule such as perlecan with a diameter of 1996). Interestingly, such domains are thought to be modu- 100–200 nm can serve to cluster extracellular domains of lators of Wnt/b- catenin and Wnt/calcium signaling, which transmembrane proteins, stabilize their interactions, and play key roles in many biological processes (Yang 2003). hence create stable “signalosomes” that can modulate cell Perlecan modulation of Wnt signaling has been reported in function. The structure and location of perlecan in the territor- Drosophila and in C. elegans (Merz et al. 2003; Nobuo ial matrix of various cells make it an ideal candidate to serve 2003), but has not been examined in mammals. Domain III as an extracellular scaffold and may provide a novel of Pln contains both laminin B and laminin EGF domains, explanation for why nature created perlecan as a long the latter of which contain repeats of about 60 amino acids complex heterofunctional-binding protein. in length that include four conserved disulfide bonds and form inflexible rod-like structures (Chakravarti et al. 1995). Summary and future directions Domain IV of Pln contains three types of immunoglobulin (Ig) domains all of which have a fold that consists of a beta- Perlecan expression changes in a dynamic fashion during pre sandwich formed of seven strands in two sheets with a and peri-implantation stage embryo development as well as in Greek-key topology (Hopf et al. 1999). Individual Ig domains decidual and placental tissues at the fetal–maternal interface. are stabilized by disulfide bonds. Such domains typically are These changes correspond with key implantation-related found in cell-adhesion domains of a variety of proteins includ- events and are consistent with the proposed roles for perlecan ing various cell adhession molecules, and are found in the extra- in promoting cell adhesion, growth factor binding, ECM organ- cellular domains of sodium channel-b subunits, and are highly ization and modulation of signaling pathways affecting cell pro- interactive in both cell–cell and cell-substratum-binding events liferation and apoptosis. Perlecan’s large size along with the (Crossin and Krushel 2000). Pln domain V, cleaved to produce distinct physical, biochemical, and biological activities of its endorepellin (Bix et al. 2004), contains a fourth alternate individual modular domains are consistent with a major 902 Perlecan—a multifunctional extracellular proteoglycan scaffold function as a macromolecule that provides a support or scaffold Carson DD, Bagchi I, Dey SK, Enders AC, Fazleabas AT, Lessey BA, for coordination of the bioactivity of multiple, complex cellular, Yoshinaga K. 2000. Embryo implantation. Dev Biol. 223:217–237. and tissue morphogenetic events. Alternative mRNA splicing Carson DD, Tang JP, Julian J. 1993. Heparan sulfate proteoglycan (perlecan) expression by mouse embryos during acquisition of attachment compe- and/or proteolytic processing would give rise to perlecan var- tence. Dev Biol. 155:97–106. iants or fragments that either are devoid of certain activities or Chaddha V, Viero S, Huppertz B, Kingdom J. 2004. Developmental biology of gain new ones, e.g. endorepellin. Future studies in the fetal–pla- the placenta and the origins of placental insufficiency. Semin Fetal cental unit should be aimed at determining to what extent Neonatal Med. 9:357–369. alternative splicing and proteolytic processing of perlecan Chakravarti S, Horchar T, Jefferson B, Laurie GW, Hassell JR. 1995. occurs in these tissues as well as the factors that control perlecan Recombinant domain III of perlecan promotes cell attachment through transcription. The critical biological importance of implantation its RGDS sequence. J Biol Chem. 270:404–409. Chen CH, HansmaHG. 2000. macromolecules: insights and placentation along with the accessibility of various models from atomic force microscopy. J Struct Biol. 131:44–55. of these processes make this an ideal system to study novel

Chen CP, Chang SC, Vivian Yang WC. 2006. High glucose alters proteogly- Downloaded from https://academic.oup.com/glycob/article/17/9/897/1987692 by guest on 29 September 2021 aspects of perlecan biology. can expression and the glycosaminoglycan composition in placentas of women with gestational diabetes mellitus and in cultured trophoblasts. Placenta. 28:97–106. Acknowledgements Chu CL, Buczek-Thomas JA, Nugent MA. 2004. Heparan sulphate proteogly- cans modulate fibroblast growth factor-2 binding through a lipid raft- The authors appreciate the careful reading and helpful com- mediated mechanism. Biochem J. 379:331–341. ments of Dr Catherine Kirn-Safran and Ms Sonia D’Souza, Cohen IR, Grassel S, Murdoch AD, Iozzo RV. 1993. Structural characteriz- who also performed some of the staining and imaging shown ation of the complete human perlecan gene and its promoter. Proc Natl in Figure 1. We are also grateful for the excellent secretarial Acad Sci USA. 90:10404–10408. assistance of Mrs Doreen Anderson and Ms Sharron Costell M, Gustafsson E, Aszodi A, Morgelin M, Bloch W, Hunziker E, Kingston. The authors were supported by NIH grants Addicks K, Timpl R, Fassler R. 1999. Perlecan maintains the integrity of cartilage and some basement membranes. J Cell Biol. 147:1109–1122. HD25235, NCI P01 CA09891.2 and, COBRE P20-RR16458. Costell M, Sasaki T, Mann K, Yamada Y, Timpl R. 1996. Structural character- ization of recombinant domain II of the basement membrane proteoglycan perlecan. FEBS Lett. 396:127–131. Conflict of interest statement Crossin KL, Krushel LA. 2000. Cellular signaling by neural cell adhesion mol- None declared. ecules of the immunoglobulin superfamily. Dev Dyn. 218:260–279. Das SK, Chakraborty I, Paria BC, Wang XN, Plowman G, Dey SK. 1995. Amphiregulin is an implantation-specific and progesterone-regulated Abbreviations gene in the mouse uterus. Mol Endocrinol. 9:691–705. Das SK, Wang XN, Paria BC, Damm D, Abraham JA, Klagsbrun M, Andrews BMP, bone morphogenetic protein; DDSH, dyssegmental dys- GK, Dey SK. 1994. Heparin-binding EGF-like growth factor gene is plasia, Silverman-Handmaker type; ECM, extracellular matrix; induced in the mouse uterus temporally by the blastocyst solely at the site of its apposition: a possible ligand for interaction with blastocyst FGFs, fibroblast growth factors; HS, heparan sulfate; HSPGs, EGF-receptor in implantation. Development. 120:1071–1083. heparan sulfate proteoglycans; LDL, low-density ; Datta MW, Hernandez AM, Schlicht MJ, Kahler AJ, DeGueme AM, Dhir R, MMP, matrix metalloproteases; SJSI, Schwartz-Jampel syn- Shah RB, Farach-Carson C, Barrett A, Datta S. 2006. Perlecan, a candidate drome type I; VEGF, vascular endothelial growth factor. gene for the CAPB , regulates prostate cancer cell growth via the sonic hedgehog pathway. Mol Cancer. 59. Dolan M, Horchar T, Rigatti B, Hassell JR. 1997. Identification of sites in References domain I of perlecan that regulate heparan sulfate synthesis. J Biol Chem. 272:4316–4322. Akerlund M. 1994. Vascularization of human endometrium. Uterine blood d’Ortho MP, Will H, Atkinson S, Butler G, Messent A, Gavrilovic J, Smith B, flow in healthy condition and in primary dysmenorrhoea. Ann N Y Acad Timpl R, Zardi L, Murphy G. 1997. Membrane-type matrix metalloprotei- Sci. 734:47–56. nases 1 and 2 exhibit broad-spectrum proteolytic capacities comparable to Aplin JD, Kimber SJ. 2004. Trophoblast-uterine interactions at implantation. many matrix metalloproteinases. Eur J Biochem. 250:751–757. Reprod Biol Endocrinol. 2:48. Dziadek M, Fujiwara S, Paulsson M, Timpl R. 1985. Immunological character- Arikawa-Hirasawa E, Watanabe H, Takami H, Hassell JR, Yamada Y. 1999. ization of basement membrane types of heparan sulfate proteoglycan. Perlecan is essential for cartilage and cephalic development. Nat Genet. Embo J. 4:905–912. 23:354–358. Edidin M. 2003. The state of lipid rafts: from model membranes to cells. Annu Arikawa-Hirasawa E, Wilcox WR, Yamada Y. 2001. Dyssegmental dysplasia, Rev Biophys Biomol Struct. 32:257–283. Silverman-Handmaker type: unexpected role of perlecan in cartilage devel- opment. Am J Med Genet. 106:254–257. Enders AC, Carter AM. 2006. Comparative placentation: some interesting modifications for histotrophic nutrition—a review. Placenta. 27:S11–16. Armant DR. 2005. Blastocysts don’t go it alone. Extrinsic signals fine-tune the intrinsic developmental program of trophoblast cells. Dev Biol. 280: Farach MC, Tang JP, Decker GL, Carson DD. 1987. Heparin/heparan sulfate 260–280. is involved in attachment and spreading of mouse embryos in vitro. Dev Bix G, Fu J, Gonzalez EM, Macro L, Barker A, Campbell S, Zutter MM, Biol. 123:401–410. Santoro SA, Kim JK, Hook M, et al. 2004. Endorepellin causes endothelial Farach-Carson MC, Hecht JT, Carson DD. 2005. Heparan sulfate proteogly- cell disassembly of actin cytoskeleton and focal adhesions through alpha2- cans: key players in cartilage biology. Crit Rev Eukaryot Gene Expr. 15: beta1 integrin. J Cell Biol. 166:97–109. 29–48. Brown JC, Sasaki T, Gohring W, Yamada Y, Timpl R. 1997. The C-terminal Fjeldstad K, Kolset SO. 2005. Decreasing the metastatic potential in cancers– domain V of perlecan promotes beta1 integrin-mediated cell adhesion, targeting the heparan sulfate proteoglycans. Curr Drug Targets. 6: binds heparin, nidogen and fibulin-2 and can be modified by glycosamino- 665–682. glycans. Eur J Biochem. 250:39–46. Fontana V, Choren V, Vauthay L, Calvo JC, Calvo L, Cameo M. 2004. Cao A, Cianchetti C, Calisti L, de Virgiliis S, Ferreli A, Tangheroni W. 1978. Exogenous interferon-gamma alters murine inner cell mass and trophoblast Schwartz-Jampel syndrome. Clinical, electrophysiological and histopatho- development. Effect on the expression of ErbB1, ErbB4 and heparan logical study of a severe variant. J Neurol Sci. 35:175–187. sulfate proteoglycan (perlecan). Reproduction. 128:717–725. 903 MC Farach-Carson and DD Carson

French MM, Smith SE, Akanbi K, Sanford T, Hecht J, Farach-Carson MC, Nicolau DV Jr, Burrage K, Parton RG, Hancock JF. 2006. Identifying optimal Carson DD. 1999. Expression of the heparan sulfate proteoglycan, perle- lipid raft characteristics required to promote nanoscale protein-protein can, during mouse embryogenesis and perlecan chondrogenic activity in interactions on the plasma membrane. Mol Cell Biol. 26:313–323. vitro. J Cell Biol. 145:1103–11015. Nobuo N. 2003. Metabolism of heparan sulfate proteoglycans in Drosophila Friedrich MV, Gohring W, Morgelin M, Brancaccio A, David G, Timpl R. cell lines. Kokubyo Gakkai Zasshi. 70:40–45. 1999. Structural basis of glycosaminoglycan modification and of heteroty- Paria BC, Ma W, Tan J, Raja S, Das SK, Dey SK, Hogan BL. 2001. Cellular pic interactions of perlecan domain V. J Mol Biol. 294:259–270. and molecular responses of the uterus to embryo implantation can be eli- Gonzalez EM, Reed CC, Bix G, Fu J, Zhang Y, Gopalakrishnan B, Greenspan cited by locally applied growth factors. Proc Natl Acad Sci USA. 98: DS, Iozzo RV. 2005. BMP-1/tolloid-like metalloproteases process endor- 1047–1052. epellin, the angiostatic C-terminal fragment of perlecan. J Biol Chem. 280: Park Y, Ng C, Datta S. 2003. Induction of string rescues the neuroblast pro- 7080–7087. liferation defect in trol mutant animals. Genesis. 36:187–195. Hayashi K, Madri JA, Yurchenco PD. 1992. Endothelial cells interact with the Park Y, Rangel C, Reynolds MM, Caldwell MC, Johns M, Nayak M, Welsh core protein of basement membrane perlecan through beta 1 and beta 3 CJ, McDermott S, Datta S. 2003. Drosophila perlecan modulates FGF integrins: an adhesion modulated by glycosaminoglycan. J Cell Biol. and hedgehog signals to activate neural stem cell division. Dev Biol. 119:945–959. 253:247–257. Downloaded from https://academic.oup.com/glycob/article/17/9/897/1987692 by guest on 29 September 2021 Hopf M, Gohring W, Kohfeldt E, Yamada Y, Timpl R. 1999. Recombinant Parr MB, Tung HN, Parr EL. 1986. The ultrastructure of the rat primary decid- domain IV of perlecan binds to nidogens, laminin-nidogen complex, fibro- ual zone. Am J Anat. 176:423–436. nectin, fibulin-2 and heparin. Eur J Biochem. 259:917–925. Raghupathy R. 2001. Pregnancy: success and failure within the Th1/Th2/Th3 Hopf M, Gohring W, Mann K, Timpl R. 2001. Mapping of binding sites for paradigm. Semin Immunol. 13:219–227. nidogens, fibulin-2, fibronectin and heparin to different IG modules of per- Rodgers KD, Sasaki T, Aszodi A, Jacenko O. 2007. Reduced perlecan in mice lecan. J Mol Biol. 311:529–541. results in chondrodysplasia resembling Schwartz-Jampel syndrome. Hum Ianoul A, Grant DD, Rouleau Y, Bani-Yaghoub M, Johnston LJ, Pezacki JP. Mol Genet. 16:515–528. 2005. Imaging nanometer domains of beta-adrenergic receptor complexes Rogalski TM, Mullen GP, Bush JA, Gilchrist EJ, Moerman DG. 2001. UNC- on the surface of cardiac myocytes. Nat Chem Biol. 1:196–202. 52/perlecan isoform diversity and function in Caenorhabditis elegans. Iozzo RV. 2005. Basement membrane proteoglycans: from cellar to ceiling. Biochem Soc Trans. 29:171–176. Nat Rev Mol Cell Biol. 6:646–656. Rohde LH, Janatpore MJ, McMaster MT, Fisher S, Zhou Y, Lim KH, French Irving-Rodgers HF, Catanzariti KD, Aspden WJ, D’Occhio MJ, Rodgers RJ. M, Hoke D, Julian J, Carson DD. 1998. Complementary expression of HIP, 2006. Remodeling of extracellular matrix at ovulation of the bovine a cell-surface heparan sulfate binding protein, and perlecan at the human ovarian follicle. Mol Reprod Dev. 73:1292–1302. fetal–maternal interface. Biol Reprod. 58:1075–1083. Iwamoto R, Yamazaki S, Asakura M, Takashima S, Hasuwa H, Miyado K, Rohde LH, Julian J, Babaknia A, Carson DD. 1996. Cell surface expression of Adachi S, Kitakaze M, Hashimoto K, Raab G, et al. 2003. Heparin- HIP, a novel heparin/heparan sulfate binding protein, of human uterine binding EGF-like growth factor and ErbB signaling is essential for heart epithelial cells and cell lines. J Biol Chem. 271:11824–11830. function. Proc Natl Acad Sci USA. 100:3221–3226. Rossi M, Morita H, Sormunen R, Airenne S, Kreivi M, Wang L, Fukai N, Jiang X, Couchman JR. 2003. Perlecan and tumor angiogenesis. J Histochem Olsen BR, Tryggvason K, Soininen R. 2003. Heparan sulfate chains of per- Cytochem. 51:1393–1410. lecan are indispensable in the lens capsule but not in the kidney. Embo J. Joseph SJ, Ford MD, Barth C, Portbury S, Bartlett PF, Nurcombe V, 22:236–245. Greferath U. 1996. A proteoglycan that activates fibroblast growth San Martin S, Soto-Suazo M, Zorn TM. 2004. Perlecan and syndecan-4 in factors during early neuronal development is a perlecan variant. uterine tissues during the early pregnancy in mice. Am J Reprod Development. 122:3443–3452. Immunol. 52:53–59. Kirn-Safran CB, Oristian DS, Focht RJ, Parker SG, Vivian JL, Carson DD. Sastry BV. 1997. Human placental cholinergic system. Biochem Pharmacol. 2007. Global growth deficiencies in mice lacking the ribosomal protein 53:1577–1586. HIP/RPL29. Dev Dyn. 236:447–460. Knox S, Whitelock J. 2006. Perlecan: how does one molecule do so many Savore C, Zhang C, Muir C, Liu R, Wyrwa J, Shu J, Zhau HE, Chung LW, things? Cell Mol Life Sci. 63:2435–2445. Carson DD, Farach-Carson MC. 2005. Perlecan knockdown in metastatic prostate cancer cells reduces heparin-binding growth factor responses in Laird SM, Tuckerman EM, Li TC. 2006. Cytokine expression in the endome- vitro and tumor growth in vivo. Clin Exp Metastasis. 22:377–390. trium of women with implantation failure and recurrent miscarriage. Reprod Biomed Online. 13:13–23. Schatz F, Krikun G, Runic R, Wang EY, Hausknecht V, Lockwood CJ. 1999. Implications of decidualization-associated protease expression in implan- Laplante P, Raymond MA, Labelle A, Abe J, Iozzo RV, Hebert MJ. 2006. tation and menstruation. Semin Reprod Endocrinol. 17:3–12. Perlecan proteolysis induces an alpha2beta1 integrin- and Src family kinase-dependent anti-apoptotic pathway in fibroblasts in the absence of Schofield KP, Gallagher JT, David G. 1999. Expression of proteoglycan core focal adhesion kinase activation. J Biol Chem. 281:30383–30392. proteins in human bone marrow stroma. Biochem J. 343:663–668. Luetteke NC, Qiu TH, Fenton SE, Troyer KL, Riedel RF, Chang A, Lee DC. Sharma B, Iozzo RV. 1998. Transcriptional silencing of perlecan gene 1999. Targeted inactivation of the EGF and amphiregulin reveals expression by interferon-gamma. J Biol Chem. 273:4642–4646. distinct roles for EGF receptor ligands in mouse mammary gland develop- Smirnov SP, Barzaghi P, McKee KK, Ruegg MA, Yurchenco PD. 2005. ment. Development. 126:2739–2750. Conjugation of LG domains of agrins and perlecan to polymerizing Melrose J, Roughley P, Knox S, Smith S, Lord M, Whitelock J. 2006. The laminin-2 promotes acetylcholine receptor clustering. J Biol Chem. 280: structure, location, and function of perlecan, a prominent pericellular pro- 41449–41457. teoglycan of fetal, postnatal, and mature hyaline cartilages. J Biol Chem. Smith SE, French MM, Julian J, Paria BC, Dey SK, Carson DD. 1997. 281:36905–36914. Expression of heparan sulfate proteoglycan (perlecan) in the mouse blas- Merz DC, Alves G, Kawano T, Zheng H, Culotti JG. 2003. UNC-52/perlecan tocyst is regulated during normal and delayed implantation. Dev Biol. affects gonadal leader cell migrations in C. elegans hermaphrodites 184:38–47. through alterations in growth factor signaling. Dev Biol. 256:173–186. Spartz AK, Herman RK, Shaw JE. 2004. Smu-2 and Smu-1, Caenorhabditis Mongiat M, Sweeney SM, San Antonio JD, Fu J, Iozzo RV. 2003. elegans homologs of mammalian spliceosome-associated proteins RED Endorepellin, a novel inhibitor of angiogenesis derived from the C termi- and fSAP57, work together to affect splice site choice. Mol Cell Biol. nus of perlecan. J Biol Chem. 278:4238–4249. 24:6811–6823. Mullen GP, Rogalski TM, Bush JA, Gorji PR, Moerman DG. 1999. Complex Spike CA, Davies AG, Shaw JE, Herman RK. 2002. MEC-8 regulates alterna- patterns of alternative splicing mediate the spatial and temporal distribution tive splicing of unc-52 transcripts in C. elegans hypodermal cells. of perlecan/UNC-52 in Caenorhabditis elegans. Mol Biol Cell. 10: Development. 129:4999–5008. 3205–3221. Stum M, Davoine CS, Fontaine B, Nicole S. 2005. Schwartz-Jampel syndrome Murray MJ, Lessey BA. 1999. Embryo implantation and tumor metastasis: and perlecan deficiency. Acta Myol. 24:89–92. common pathways of invasion and angiogenesis. Semin Reprod Stum M, Davoine CS, Vicart S, Guillot-Noel L, Topaloglu H, Carod-Artal FJ, Endocrinol. 17:275–290. Kayserili H, Hentati F, Merlini L, Urtizberea JA, et al. 2006. Spectrum of 904 Perlecan—a multifunctional extracellular proteoglycan scaffold

HSPG2 (perlecan) mutations in patients with Schwartz-Jampel syndrome. Voigt A, Pflanz R, Schafer U, Jackle H. 2002. Perlecan participates in proliferation Hum Mutat. 27:1082–1091. activation of quiescent Drosophila neuroblasts. Dev Dyn. 224:403–412. Sugaya K, Hongo E, Ishihara Y, Tsuji H. 2006. The conserved role of Smu1 in Whitelock JM, Murdoch AD, Iozzo RV, Underwood PA. 1996. The degra- splicing is characterized in its mammalian temperature-sensitive mutant. dation of human endothelial cell-derived perlecan and release of bound J Cell Sci. 119:4944–4951. basic fibroblast growth factor by stromelysin, collagenase, plasmin, and Takamoto N, Zhao B, Tsai SY, DeMayo FJ. 2002. Identification of Indian heparanases. J Biol Chem. 271:10079–10086. hedgehog as a progesterone-responsive gene in the murine uterus. Mol Yang WC, Su TH, Yang YC, Chang SC, Chen CY, Chen CP. 2005. Altered Endocrinol. 16:2338–2348. perlecan expression in placental development and gestational diabetes mel- Thadikkaran L, Crettaz D, Siegenthaler MA, Gallot D, Sapin V, Iozzo RV, litus. Placenta. 26:780–788. Queloz PA, Schneider P, Tissot JD. 2005. The role of proteomics in the Yang Y. 2003. Wnts and wing: Wnt signaling in vertebrate limb development assessment of premature rupture of fetal membranes. Clin Chim Acta. and musculoskeletal morphogenesis. Birth Defects Res C Embryo Today. 360:27–36. 69:305–317. Downloaded from https://academic.oup.com/glycob/article/17/9/897/1987692 by guest on 29 September 2021

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