Type-X collagen interacts with the small leucine-rich proteoglycans decorin and biglycan

Introduction Type‐X collagen is expressed by hypertrophic chondrocytes in the epiphyseal growth plate. The 59‐kDa α‐chain consists of a 45‐kDa triple‐helical domain flanked by two noncollagenous regions, a large C‐terminal domain termed NC1 and a smaller N‐terminal domain termed NC2. The restricted distribution of type‐X collagen within the growth plate indicates a potential role during the process of endochondral ossification. Type‐X collagen may form a hexagonal lattice‐like matrix, permissive to vascular invasion and mineralization. Decorin and biglycan are small leucine‐rich proteoglycans, which are usually substituted with one or two glycosaminoglycan (GAG) chains, respectively. Their 40‐kDa protein cores contain N‐terminal GAG attachment site(s), several central leucine‐rich repeats and a disulphide‐bonded loop at the C‐terminal. They are ubiquitously expressed and are found in many connective tissues, including skin, cartilage and bone. They are known to interact with many proteins including fibrillar collagens. The molecular interactions of type‐X collagen with decorin and biglycan have been investigated in vitro. Characterizing these interactions may elucidate the precise role of these complexes in the hypertrophic cartilage matrix. Materials and methods To investigate the interactions of type‐X collagen with decorin and biglycan, solid phase assays, including competitive assays and surface plasmon resonance were used. Proteins used during the investigation included type‐X collagen purified from embryonic chick tibial hypertrophic chondrocytes, pepsin‐treated type‐X collagen, human recombinant NC1 domain of type‐X collagen, human recombinant decorin and biglycan purified from bovine cartilage. Results Type‐X collagen interacts with biglycan and decorin in solid phase assays and surface plasmon resonance, using the BIAcore 3000 system. The interactions occur primarily via the NC1 domain of type‐X collagen and are not dependent on the presence of the GAG chains on the proteoglycans. Dissociation constants have been calculated and indicate high affinity binding. Results from competitive binding assays indicate that decorin and biglycan bind to the same site on type‐X collagen. Rotary shadowing is currently being used to confirm interactions and to locate the interaction sites better. Discussion Interactions between type‐X collagen and other matrix components may be required for the assembly of the hypertrophic cartilage matrix and to maintain its integrity. Within the growth plate, type‐X collagen interactions with decorin and biglycan may have potential roles in regulation or maintenance of the type‐X collagen hexagonal network and/or presentation of growth factors, e.g. TGF‐β known to be important in endochondral ossification.     

Immunoglobulin superfamily receptors in protochordates: before RAG time

Summary:  Urochordates and cephalochordates do not have an adaptive immune system involving the somatic rearrangement of their antigen receptor genes. They do not have antigen-presenting molecules of the major histocompatibility complex (MHC)-linked class I and II types. In the absence of such a system, the status of their genes reflects perhaps a primitive pre-recombination-activating gene (RAG) stage that could suggest the pathway leading to the genesis of the T-cell receptor (TCR) and antibodies.
In the genome of Ciona intestinalis , genes that encode molecules with membrane receptor features have been found among many members of the immunoglobulin superfamily (Igsf). They use the domains typical of vertebrate antigen receptors and class I and II: the V, and C1-like domains. These genes belong to two families with recognizable homologs in vertebrates: the junctional adhesion molecule (JAM)/cortical thymocyte marker of Xenopus (CTX) family and the nectin family. The human homologs of these genes segregate in a single unit of four paralogous segments on chromosomes 1q, 3q, 11p, and 21q. These regions contain nowadays several genes involved in the adaptive immune system, and some related members are present in the MHC paralogs as well. They also contain receptor-like genes without homologs in Ciona but with related members in the protostome Drosophila .
It looks as if in Ciona one detects what looks like the 'fossil' of one group of genes bound to duplicate and give rise to many crucial elements of the adaptive immune system. The modern homologs of these JAM, CTX, and nectins are all or almost all virus receptors, and the hypothesis is formulated that this property was taken advantage of during evolution to participate in the elaboration of either or both the somatically generated antigen-recognizing receptors and the antigen-presenting molecules.     

C1q and tumor necrosis factor superfamily: modularity and versatility

C1q is the target recognition protein of the classical complement pathway and a major connecting link between innate and acquired immunity. As a charge pattern recognition molecule of innate immunity, C1q can engage a broad range of ligands via its globular (gC1q) domain and modulate immune cells, probably via its collagen region. The gC1q signature domain, also found in many non-complement proteins, has a compact jelly-roll beta-sandwich fold similar to that of the multifunctional tumor necrosis factor (TNF) ligand family. The members of this newly designated 'C1q and TNF superfamily' are involved in processes as diverse as host defense, inflammation, apoptosis, autoimmunity, cell differentiation, organogenesis, hibernation and insulin-resistant obesity. This review is an attempt to draw structural and functional parallels between the members of the C1q and TNF superfamily.     

C1q and its growing family

C1q is the target recognition protein of the classical complement pathway and a major connecting link between innate and acquired immunity. As a charge pattern recognition molecule of innate immunity, C1q can engage a broad range of self and non-self ligands via its heterotrimeric globular (gC1q) domain and thus trigger the classical pathway. The trimeric gC1q signature domain has been identified in a variety of non-complement proteins that can be grouped together as a C1q family. The X-ray crystal structures of the gC1q domain of a few members of the C1q family reveal a compact jelly-roll beta-sandwich fold similar to that of the multifunctional tumor necrosis factor (TNF) ligand family, hence the C1q and TNF superfamily. This review is an update on the structural and functional aspects of the gC1q domain of human C1q. We also mention the diverse range of proteins that utilize a gC1q domain in order to reflect on its importance as a versatile scaffold to support a variety of functions.     

KH domain-containing proteins of yeast: absence of a fragile X gene homologue.

The KH domain is a region defined by its homology to the RNA-binding domains of the heterogeneous nuclear ribonucleoprotein K (hnRNPK). There are two such domains in the FMR1 protein which is underexpressed in the fragile X syndrome. We developed a computer method to search the S. cerevisiae protein sequences as they became available for the KH domain of the FMR1 protein. Using our motif and FINDPATTERNS of the Wisconsin Package of GCG, nine proteins were identified in the completed yeast ORF database that contain KH domains. Five proteins have known or predicted functions; four await functional analysis. Using GeneWorks and GeneJockeyII alignments, we found that the yeast protein KH domain showing the most similarity to either FMR1P KH domain was a KH domain in HX/SCP160. Its sequence is 50% identical to the second KH domain of FMR1P. However, SCP160 contains eight conserved and six degenerate KH domains. Further analysis showed that SCP160 is a better match overall to the vertebrate and C. elegans protein Vigilin, which also contains 14 KH domains. The next most similar yeast KH domain was found in YB83, a protein shorter than FMR1P and containing three KH domains, one of which shares 45% identity with the second KH domain in FMR1P. There is no significant overall sequence similarity between this yeast protein and FMR1P. Thus, while several proteins in yeast contain KH domains, no apparent yeast homologue exists for the FMR1 protein of the fragile X gene family.     

Identification and functional characterization of imatinib‐sensitive DTD1‐PDGFRB and CCDC88C‐PDGFRB fusion genes in eosinophilia‐associated myeloid/lymphoid neoplasms

Eosinophilia‐associated myeloid neoplasms with rearrangement of chromosome bands 5q31‐33 are frequently associated with PDGFRB fusion genes, which are exquisitely sensitive to treatment with imatinib. In search for novel fusion partners of PDGFRB, we analyzed three cases with translocation t(5;20)(q33;p11), t(5;14)(q33;q32), and t(5;17;14)(q33;q11;q32) by 5′‐rapid amplification of cDNA ends polymerase chain reaction (5′‐RACE‐PCR) and DNA‐based long‐distance inverse PCR (LDI‐PCR) with primers derived from PDGFRB. LDI‐PCR revealed a fusion between CCDC88C exon 25 and PDGFRB exon 11 in the case with t(5;17;14)(q33;q11;q32) while 5′‐RACE‐PCR identified fusions between CCDC88C exon 10 and PDGFRB exon 12 and between DTD1 exon 4 and PDGFRB exon 12 in the cases with t(5;14)(q33;q32) and t(5;20)(q33;p11), respectively. The PDGFRB tyrosine‐kinase domain is predicted to be retained in all three fusion proteins. The partner proteins contained coiled‐coil domains or other domains, which putatively lead to constitutive activation of the PDGFRB fusion protein. In vitro functional analyses confirmed transforming activity and imatinib‐sensitivity of the fusion proteins. All three patients achieved rapid and durable complete hematologic remissions on imatinib. © 2014 Wiley Periodicals, Inc.     

PRG‐4/SZP N‐ and C‐terminal domains: cloning,expression and characterization

Introduction Proteoglycan‐4 (PRG‐4), also known as superficial zone protein/proteoglycan (SZP), is an approximately 345‐kDa mucinous proteoglycan that has been detected in a variety of tissues including cartilage, tendon, bone, heart and liver ( Ikegawa et al. 2000 ). In the synovial joint, PRG‐4 is specifically synthesized by chondrocytes located in the superficial zone of articular cartilage and by some surface‐lining cells of the synovium ( Schumacher et al. 1994 ). Sequence analyses have shown that the N‐ and C‐terminal vitronectin‐like domains of PRG‐4 may impart interesting functions relevant to synovial joint metabolism ( Merberg et al. 1993 ; Flannery et al. 1999 ). The objective of this study was to investigate these potential functions, facilitated by the production of PRG‐4 N‐ and C‐terminal domains as recombinant proteins. Methods cDNAs for the human N‐terminal (exons 2–5) and bovine C‐terminal (exons 7–12) domains of PRG‐4 were obtained by RT‐PCR and cloned into the expression vector pMT‐BiP for inducible, secreted expression in Drosophila S2 cells. Proteins were purified using FLAG‐M2 antibody affinity chromatography and visualized by SDS‐PAGE and Western blotting with PRG‐4‐specific antibodies. The heparin‐binding properties of recombinant proteins were investigated using heparin affinity chromatography. The interactions of recombinant PRG‐4 domains with human plasminogen activator‐inhibitor (PAI)‐1 and bovine type‐II collagen were assayed using standard ELISA techniques. Results Stable cell lines have been generated that express human N‐terminal and bovine C‐terminal PRG‐4 domains. In both cases, two proteins have been purified, possibly due to a splice mechanism by the expression system. N‐terminal sequence data and Western blotting indicate that the two species in each case could represent full‐length and truncated proteins. Analyses of the two PRG‐4 N‐terminal domain species have confirmed the presence of a predicted heparin‐binding domain and indicate that the molecule can bind to PAI‐1, with binding activity localized towards its two somatomedin B domains. The somatomedin B domain of vitronectin is known to bind PAI‐1 ( Seiffert 1997 ). Analyses of the two PRG‐4 C‐terminal species have demonstrated self‐association under nonreducing conditions and binding to heparin and PAI‐1. Discussion The exact role of PRG‐4 in the synovial joint is yet to be elucidated. However, these results point towards the interaction of the N‐ and C‐terminal domains of PRG‐4 with structural molecules such as type‐II collagen and heparin, and functional molecules such as PAI‐1, a serpin that is involved in the fibrinolytic cascade and cell adhesion. These properties are in addition to the well‐documented boundary lubricating activity of the central mucinous region of PRG‐4.     

Partial protein sequence of the globular domain of alpha 4(IV) collagen chain: sites of sequence variability and homology with alpha 2(IV).

The globular domain (NC) of alpha 4(IV) collagen chain was partially sequenced and compared with the NC domain of other collagen IV chains. The alpha 4(IV) NC domain was found to be most closely related to alpha 2(IV) NC domain but distinct from the NC domain of alpha 1(IV), alpha 2(IV), alpha 3(IV) and alpha 5(IV) collagen chains. Partial sequence, representing nearly one half of alpha 4(IV) NC domain, shows 56%, 69%, 51% and 54% identity with the corresponding NC domains of alpha 1(IV), alpha 2(IV), alpha 3(IV) and alpha 5(IV) collagen chains, respectively. A short, highly polar, region of variable sequence is found near the carboxy terminus of alpha 4(IV) NC domain. This sequence corresponds to a non-conserved region among NC domains, suggesting functional specialization at this site. It exhibits high surface probability with predicted structural differences among NC domains. These results confirm uniqueness of alpha 4(IV) NC domain and indicate its structural relatedness to other NC domains of collagen IV.     

Fusion of PDGFRB to MPRIP,CPSF6, and GOLGB1 in three patients with eosinophilia‐associated myeloproliferative neoplasms

In eosinophilia‐associated myeloproliferative neoplasms (MPN‐eo), constitutive activation of protein tyrosine kinases (TK) as consequence of translocations, inversions, or insertions and creation of TK fusion genes is recurrently observed. The most commonly involved TK and their potential TK inhibitors include PDGFRA at 4q12 or PDGFRB at 5q33 (imatinib), FGFR1 at 8p11 (ponatinib), and JAK2 at 9p24 (ruxolitinib). We here report the identification of three new PDGFRB fusion genes in three male MPN‐eo patients: MPRIP‐PDGFRB in a case with t(5;17)(q33;p11), CPSF6‐PDGFRB in a case with t(5;12)(q33;q15), and GOLGB1‐PDGFRB in a case with t(3;5)(q13;q33). The fusion proteins identified by 5′‐rapid amplification of cDNA ends polymerase chain reaction (PCR) or DNA‐based long distance inverse PCR are predicted to contain the TK domain of PDGFRB. The partner genes contain domains like coiled‐coil structures, which are likely to cause dimerization and activation of the TK. In all patients, imatinib induced rapid and durable complete remissions. © 2015 Wiley Periodicals, Inc.     

Fc receptor homologs: newest members of a remarkably diverse Fc receptor gene family

Summary: Newfound relatives of the classical Fc receptors (FcR) have been provisionally named the Fc receptor homologs (FcRH). The recent identification of eight human and six mouse FcRH genes substantially increases the size and functional potential of the FcR family. The extended family of FcR and FcRH genes spans ～15 Mb of the human chromosome 1q21–23 region, whereas in mice this family is split between chromosomes 1 and 3. The FcRH genes encode molecules with variable combinations of five subtypes of immunoglobulin (Ig) domains. The presence of a conserved sequence motif in one Ig domain subtype implies Ig Fc binding capability for many FcRH family members that are preferentially expressed by B lineage cells. In addition, most FcRH family members have consensus tyrosine‐based activating and inhibitory motifs in their cytoplasmic domains, while the others lack features typical of transmembrane receptors. The FcRH family members, like the classical FcRs, come in multiple isoforms and allelic variations. The unique individual and polymorphic properties of the FcR/FcRH members indicate a remarkably diverse Fc receptor gene family with immunoregulatory function.     

Clinical and molecular genetic characterization of two female patients harboring the Xq27.3q28 deletion with different ratios of X chromosome inactivation

A heterozygous deletion at Xq27.3q28 including FMR1, AFF2, and IDS causing intellectual disability and characteristic facial features is very rare in females, with only 10 patients having been reported. Here, we examined two female patients with different clinical features harboring the Xq27.3q28 deletion and determined the chromosomal breakpoints. Moreover, we assessed the X chromosome inactivation (XCI) in peripheral blood from both patients. Both patients had an almost overlapping deletion at Xq27.3q28, however, the more severe patient (Patient 1) showed skewed XCI of the normal X chromosome (79:21) whereas the milder patient (Patient 2) showed random XCI. Therefore, deletion at Xq27.3q28 critically affected brain development, and the ratio of XCI of the normal X chromosome greatly affected the clinical characteristics of patients with deletion at Xq27.3q28. As the chromosomal breakpoints were determined, we analyzed a change in chromatin domains termed topologically associated domains (TADs) using published Hi‐C data on the Xq27.3q28 region, and found that only patient 1 had a possibility of a drastic change in TADs. The altered chromatin topologies on the Xq27.3q28 region might affect the clinical features of patient 1 by changing the expression of genes just outside the deletion and/or the XCI establishment during embryogenesis resulting in skewed XCI.     

Prediction of the Repeat Domain Structures and Impact of Parkinsonism‐Associated Variations on Structure and Function of all Functional Domains of Leucine‐Rich Repeat Kinase 2 (LRRK2)

Genetic variations of leucine‐rich repeat kinase 2 (LRRK2) are the major cause of dominantly inherited Parkinson disease (PD). LRRK2 protein contains seven predicted domains: a tandem Ras‐like GTPase (ROC) domain and C‐terminal of Roc (COR) domain, a protein kinase domain, and four repeat domains. PD‐causative variations arise in all domains, suggesting that aberrant functioning of any domain can contribute to neurotoxic mechanisms of LRRK2. Determination of the three‐dimensional structure of LRRK2 is one of the best avenues to decipher its neurotoxic mechanism. However, with the exception of the Roc domain, the three‐dimensional structures of the functional domains of LRRK2 have yet to be determined. Based on the known three‐dimensional structures of repeat domains of other proteins, the tandem Roc–COR domains of the Chlorobium tepidum Rab family protein, and the kinase domain of the Dictyostelium discoideum Roco4 protein, we predicted (1) the motifs essential for protein–protein interactions in all domains, (2) the motifs critical for catalysis and substrate recognition in the tandem Roc–COR and kinase domains, and (3) the effects of some PD‐associated missense variations on the neurotoxic action of LRRK2. Results of our analysis provide a conceptual framework for future investigation into the regulation and the neurotoxic mechanism of LRRK2.     

Novel missense mutation resulting in the substitution of tyrosine by cysteine at codon 597 of the type X collagen gene associated with Schmid metaphyseal chondrodysplasia

Schmid metaphyseal chondrodysplasia (SMCD) is one of the most common forms of the osteochondrodysplasias. Mutations or deletions in the COL10A1 gene that encodes type X collagen have been shown to cause this disorder. Most of the gene mutations and deletions are located in the non-collagenous carboxy (C)-terminal (NC1) domain. We describe a novel missense mutation in a patient with SMCD that leads to the substitution of Tyr at codon 597 by Cys in the NC1 domain. Sequence analysis indicated that the proband was heterozygous for the mutation. Her parents were homozygous for the normal sequence, indicating the de-novo occurrence of this mutation. Received: April 27, 1998 / Accepted: June 24, 1998     

Homozygous Gly530Ser substitution in COL5A1 causes mild classical Ehlers‐Danlos syndrome

Skin hyperelasticity, tissue fragility with atrophic scars, and joint hypermobility are characteristic for the classical type of Ehlers‐Danlos syndrome (EDS). The disease is usually inherited as an autosomal dominant trait; however, recessive mode of inheritance has been documented in tenascin‐X‐deficient EDS patients. Mutations in the genes coding for collagen α1(V) chain (COL5A1), collagen α2(V) chain (COL5A2), tenascin‐X (TNX), and collagen α1(I) chain (COL1A1) have been characterized in patients with classical EDS, thus confirming the suspected genetic heterogeneity. Recently, we described a patient with severe classical EDS due to a Gly1489Glu substitution in the α1(V) triple‐helical domain who was, in addition, heterozygous for a disease‐modifying Gly530Ser substitution in the α1(V) NH₂‐terminal domain [Giunta and Steinmann, 2000 : Am. J. Med. Genet. 90:72–79; Steinmann and Giunta, 2000 : Am. J. Med. Genet. 93:342]. Here, we report on a 4‐year‐old boy with mild classical EDS, born to healthy consanguineous Turkish parents; the mother presented a soft skin, while the father had a normal thick skin. Ultrastructural analysis of the dermis revealed in the patient the typical “cauliflower” collagen fibrils, while in both parents variable moderate aberrations were seen. Mutation revealed the presence of a homozygous Gly530Ser substitution in the α1(V) collagen chains in the patient, while both parents were heterozygous for the same substitution. An additional mutation in either the COL5A1 and COL5A2 genes was excluded. Furthermore, haplotype analysis with polymorphic microsatellite markers excluded linkage to the genes coding for α3(V) collagen (COL5A3), tenascin‐X (TNX), thrombospondin‐2 (THBS2), and decorin (DCN). These new findings support further our previous hypothesis that the heterozygous Gly530Ser substitution is disease modifying and now suggest that in the homozygous state it is disease causing. © 2002 Wiley‐Liss, Inc.     

Missense mutation in a von Willebrand factor type A domain of the alpha 3(VI) collagen gene (COL6A3) in a family with Bethlem myopathy

The Bethlem myopathy is a rare autosomal dominant proximal myopathy characterized by early childhood onset and joint contractures. Evidence for linkage and genetic heterogeneity has been established, with the majority of families linked to 21q22.3 and one large family linked to 2q37, implicating the three type VI collagen subunit genes, COL6A1 (chromosome 21), COL6A2 (chromosome 21) and COL6A3 (chromosome 2) as candidate genes. Mutations of the invariant glycine residues in the triple-helical domain-coding region of COL6A1 and COL6A2 have been reported previously in the chromosome 21-linked families. We report here the identification of a G-->A mutation in the N-terminal globular domain-coding region of COL6A3 in a large American pedigree (19 affected, 12 unaffected), leading to the substitution of glycine by glutamic acid in the N2 motif, which is homologous to the type A domains of the von Willebrand factor. This mutation segregated to all affected family members, to no unaffected family members, and was not identified in 338 unrelated Caucasian control chromosomes. Thus mutations in either the triple-helical domain or the globular domain of type VI collagen appear to cause Bethlem myopathy.      

Comparative Analysis of Apoptosis and Inflammation Genes of Mice and Humans

Apoptosis (programmed cell death) plays important roles in many facets of normal mammalian physiology. Host-pathogen interactions have provided evolutionary pressure for apoptosis as a defense mechanism against viruses and microbes, sometimes linking apoptosis mechanisms with inflammatory responses through NFκB induction. Proteins involved in apoptosis and NFκB induction commonly contain evolutionarily conserved domains that can serve as signatures for identification by bioinformatics methods. Using a combination of public (NCBI) and private (RIKEN) databases, we compared the repertoire of apoptosis and NFκB-inducing genes in humans and mice from cDNA/EST/genomic data, focusing on the following domain families: (1) Caspase proteases; (2) Caspase recruitment domains (CARD); (3) Death Domains (DD); (4) Death Effector Domains (DED); (5) BIR domains of Inhibitor of Apoptosis Proteins (IAPs); (6) Bcl-2 homology (BH) domains of Bcl-2 family proteins; (7) Tumor Necrosis Factor (TNF)-family ligands; (8) TNF receptors (TNFR); (9) TIR domains; (10) PAAD (PYRIN; PYD, DAPIN); (11) nucleotide-binding NACHT domains; (12) TRAFs; (13) Hsp70-binding BAG domains; (14) endonuclease-associated CIDE domains; and (15) miscellaneous additional proteins. After excluding redundancy due to alternative splice forms, sequencing errors, and other considerations, we identified cDNAs derived from a total of 227 human genes among these domain families. Orthologous murine genes were found for 219 (96%); in addition, several unique murine genes were found, which appear not to have human orthologs. This mismatch may be due to the still fragmentary information about the mouse genome or genuine differences between mouse and human repertoires of apoptotic genes. With this caveat, we discuss similarities and differences in human and murine genes from these domain families.