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1.
J Biol Chem ; 299(12): 105459, 2023 Dec.
Article in English | MEDLINE | ID: mdl-37977222

ABSTRACT

The collagen IVα345 (Col-IVα345) scaffold, the major constituent of the glomerular basement membrane (GBM), is a critical component of the kidney glomerular filtration barrier. In Alport syndrome, affecting millions of people worldwide, over two thousand genetic variants occur in the COL4A3, COL4A4, and COL4A5 genes that encode the Col-IVα345 scaffold. Variants cause loss of scaffold, a suprastructure that tethers macromolecules, from the GBM or assembly of a defective scaffold, causing hematuria in nearly all cases, proteinuria, and often progressive kidney failure. How these variants cause proteinuria remains an enigma. In a companion paper, we found that the evolutionary emergence of the COL4A3, COL4A4, COL4A5, and COL4A6 genes coincided with kidney emergence in hagfish and shark and that the COL4A3 and COL4A4 were lost in amphibians. These findings opened an experimental window to gain insights into functionality of the Col-IVα345 scaffold. Here, using tissue staining, biochemical analysis and TEM, we characterized the scaffold chain arrangements and the morphology of the GBM of hagfish, shark, frog, and salamander. We found that α4 and α5 chains in shark GBM and α1 and α5 chains in amphibian GBM are spatially separated. Scaffolds are distinct from one another and from the mammalian Col-IVα345 scaffold, and the GBM morphologies are distinct. Our findings revealed that the evolutionary emergence of the Col-IVα345 scaffold enabled the genesis of a compact GBM that functions as an ultrafilter. Findings shed light on the conundrum, defined decades ago, whether the GBM or slit diaphragm is the primary filter.


Subject(s)
Collagen Type IV , Glomerular Basement Membrane , Mammals , Animals , Anura , Collagen Type IV/classification , Collagen Type IV/genetics , Collagen Type IV/metabolism , Glomerular Basement Membrane/chemistry , Glomerular Basement Membrane/metabolism , Glomerular Basement Membrane/physiology , Hagfishes , Mammals/genetics , Mammals/metabolism , Mammals/physiology , Sharks , Species Specificity , Urodela
2.
J Biol Chem ; 299(11): 105318, 2023 11.
Article in English | MEDLINE | ID: mdl-37797699

ABSTRACT

Collagen IV scaffold is a primordial innovation enabling the assembly of a fundamental architectural unit of epithelial tissues-a basement membrane attached to polarized cells. A family of six α-chains (α1 to α6) coassemble into three distinct protomers that form supramolecular scaffolds, noted as collagen IVα121, collagen IVα345, and collagen IVα121-α556. Chloride ions play a pivotal role in scaffold assembly, based on studies of NC1 hexamers from mammalian tissues. First, Cl- activates a molecular switch within trimeric NC1 domains that initiates protomer oligomerization, forming an NC1 hexamer between adjoining protomers. Second, Cl- stabilizes the hexamer structure. Whether this Cl--dependent mechanism is of fundamental importance in animal evolution is unknown. Here, we developed a simple in vitro method of SDS-PAGE to determine the role of solution Cl- in hexamer stability. Hexamers were characterized from 34 animal species across 15 major phyla, including the basal Cnidarian and Ctenophora phyla. We found that solution Cl- stabilized the quaternary hexamer structure across all phyla except Ctenophora, Ecdysozoa, and Rotifera. Further analysis of hexamers from peroxidasin knockout mice, a model for decreasing hexamer crosslinks, showed that solution Cl- also stabilized the hexamer surface conformation. The presence of sufficient chloride concentration in solution or "chloride pressure" dynamically maintains the native form of the hexamer. Collectively, our findings revealed that chloride pressure on the outside of cells is a primordial innovation that drives and maintains the quaternary and conformational structure of NC1 hexamers of collagen IV scaffolds.


Subject(s)
Chlorides , Collagen Type IV , Animals , Mice , Protein Subunits/analysis , Protein Structure, Tertiary , Collagen Type IV/chemistry , Basement Membrane , Mammals
3.
bioRxiv ; 2023 Oct 21.
Article in English | MEDLINE | ID: mdl-37905027

ABSTRACT

Collagen IV is a primordial component of basement membranes, a specialized form of extracellular matrix that enabled multi-cellular epithelial tissues. In mammals, collagen IV assembles from a family of six α-chains (α1 to α6), encoded by six genes (COL4A1 to COL4A6), into three distinct scaffolds: the α121, the α345 and a mixed scaffold containing both α121 and α565. The six mammalian COL4A genes occur in pairs that occur in a head-to-head arrangement on three distinct chromosomes. In Alport syndrome, variants in the COL4A3, 4 or 5 genes cause either loss or defective assembly of the collagen IV α345 scaffold which results in a dysfunctional glomerular basement membrane, proteinuria and progression to renal failure in millions of people worldwide. Here, we determine the evolutionary emergence and diversification of the COL4A genes using comparative genomics and biochemical analyses. Using syntenic relationships to genes closely linked to the COL4A genes, we determine that the COL4A3 and COL4A4 gene pair appeared in cyclostomes (hagfish and lampreys) while the COL4A5 and COL4A6 gene pair emerged in gnathostomes, jawed vertebrates. The more basal chordate species, lancelets and tunicates, do not have discrete kidneys and have a single COL4A gene pair, though often with single isolated COL4 genes similar to those found in C elegans . Remarkably, while the six COL4A genes are conserved in vertebrates, amphibians have lost the COL4A3 and COL4A4 genes. Our findings of the evolutionary emergence of these genes, together with the amphibian double-knockout, opens an experimental window to gain insights into functionality of the Col IV α345 scaffold.

4.
J Immunol ; 211(6): 1042-1051, 2023 09 15.
Article in English | MEDLINE | ID: mdl-37540118

ABSTRACT

Gnathostome adaptive immunity is defined by the Ag receptors, Igs and TCRs, and the MHC. Cartilaginous fish are the oldest vertebrates with these adaptive hallmarks. We and others have unearthed nonrearranging Ag receptor-like genes in several vertebrates, some of which are encoded in the MHC or in MHC paralogous regions. One of these genes, named UrIg, was detected in the class III region of the shark MHC that encodes a protein with typical V and C domains such as those found in conventional Igs and TCRs. As no transmembrane region was detected in gene models or cDNAs, the protein does not appear to act as a receptor. Unlike some other shark Ig genes, the UrIg V region shows no evidence of RAG-mediated rearrangement, and thus it is likely related to other V genes that predated the invasion of the RAG transposon. The UrIg gene is present in all elasmobranchs and evolves conservatively, unlike Igs and TCRs. Also, unlike Ig/TCR, the gene is not expressed in secondary lymphoid tissues, but mainly in the liver. Recombinant forms of the molecule form disulfide-linked homodimers, which is the form also detected in many shark tissues by Western blotting. mAbs specific for UrIg identify the protein in the extracellular matrix of several shark tissues by immunohistochemistry. We propose that UrIg is related to the V gene invaded by the RAG transposon, consistent with the speculation of emergence of Ig/TCR within the MHC or proto-MHC.


Subject(s)
Antibodies , Major Histocompatibility Complex , Sharks , Sharks/genetics , Sharks/metabolism , Antibodies/chemistry , Antibodies/genetics , Antibodies/metabolism , Immunoglobulin G/genetics , Phylogeny , Evolution, Molecular , Amino Acid Sequence , Sequence Alignment , Liver/metabolism , Gene Expression , Mammals/genetics , Organ Specificity
5.
Curr Opin Nephrol Hypertens ; 31(3): 213-220, 2022 05 01.
Article in English | MEDLINE | ID: mdl-35283436

ABSTRACT

PURPOSE OF REVIEW: In Alport syndrome, over 1,700 genetic variants in the COL4A3, COL4A4, and COL4A5 genes cause the absence or malfunctioning of the collagen IVα345 scaffold - an essential component of the glomerular basement membrane (GBM). Therapies are limited to treatment with Angiotensin-Converting enzyme (ACE) inhibitors to slow progression of the disease. Here, we review recent progress in therapy development to replace the scaffold or restore its function. RECENT FINDINGS: Multiple approaches emerged recently for development of therapies that target different stages of production and assembly of the collagen IVα345 scaffold in the GBM. These approaches are based on (1) recent advances in technologies allowing to decipher pathogenic mechanisms that underlie scaffold assembly and dysfunction, (2) development of DNA editing tools for gene therapy, (3) RNA splicing interference, and (4) control of mRNA translation. SUMMARY: There is a growing confidence that these approaches will ultimately provide cure for Alport patients. The development of therapy will be accelerated by studies that provide a deeper understanding of mechanisms that underlie folding, assembly, and function of the collagen IVα345 scaffold.


Subject(s)
Nephritis, Hereditary , Collagen Type IV/genetics , Female , Glomerular Basement Membrane , Humans , Male , Nephritis, Hereditary/genetics , Nephritis, Hereditary/therapy , Prospective Studies
6.
J Biol Chem ; 296: 100590, 2021.
Article in English | MEDLINE | ID: mdl-33774048

ABSTRACT

Diseases of the glomerular basement membrane (GBM), such as Goodpasture's disease (GP) and Alport syndrome (AS), are a major cause of chronic kidney failure and an unmet medical need. Collagen IVα345 is an important architectural element of the GBM that was discovered in previous research on GP and AS. How this collagen enables GBM to function as a permselective filter and how structural defects cause renal failure remain an enigma. We found a distinctive genetic variant of collagen IVα345 in both a familial GP case and four AS kindreds that provided insights into these mechanisms. The variant is an 8-residue appendage at the C-terminus of the α3 subunit of the α345 hexamer. A knock-in mouse harboring the variant displayed GBM abnormalities and proteinuria. This pathology phenocopied AS, which pinpointed the α345 hexamer as a focal point in GBM function and dysfunction. Crystallography and assembly studies revealed underlying hexamer mechanisms, as described in Boudko et al. and Pedchenko et al. Bioactive sites on the hexamer surface were identified where pathogenic pathways of GP and AS converge and, potentially, that of diabetic nephropathy (DN). We conclude that the hexamer functions include signaling and organizing macromolecular complexes, which enable GBM assembly and function. Therapeutic modulation or replacement of α345 hexamer could therefore be a potential treatment for GBM diseases, and this knock-in mouse model is suitable for developing gene therapies.


Subject(s)
Anti-Glomerular Basement Membrane Disease/genetics , Collagen Type IV/genetics , Collagen Type IV/metabolism , Mutation , Nephritis, Hereditary/genetics , Animals , Collagen Type IV/chemistry , Mice , Models, Molecular , Protein Multimerization , Protein Structure, Quaternary , Signal Transduction
7.
Front Oncol ; 11: 601957, 2021.
Article in English | MEDLINE | ID: mdl-33708626

ABSTRACT

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood with a propensity to metastasize. Current treatment for patients with RMS includes conventional systemic chemotherapy, radiation therapy, and surgical resection; nevertheless, little to no improvement in long term survival has been achieved in decades-underlining the need for target discovery and new therapeutic approaches to targeting tumor cells or the tumor microenvironment. To evaluate cross-species sarcoma extracellular matrix production, we have used murine models which feature knowledge of the myogenic cell-of-origin. With focus on the RMS/undifferentiated pleomorphic sarcoma (UPS) continuum, we have constructed tissue microarrays of 48 murine and four human sarcomas to analyze expression of seven different collagens, fibrillins, and collagen-modifying proteins, with cross-correlation to RNA deep sequencing. We have uncovered that RMS produces increased expression of type XVIII collagen alpha 1 (COL18A1), which is clinically associated with decreased long-term survival. We have also identified significantly increased RNA expression of COL4A1, FBN2, PLOD1, and PLOD2 in human RMS relative to normal skeletal muscle. These results complement recent studies investigating whether soft tissue sarcomas utilize collagens, fibrillins, and collagen-modifying enzymes to alter the structural integrity of surrounding host extracellular matrix/collagen quaternary structure resulting in improved ability to improve the ability to invade regionally and metastasize, for which therapeutic targeting is possible.

8.
Adv Exp Med Biol ; 21: 129-141, 2021.
Article in English | MEDLINE | ID: mdl-32979156

ABSTRACT

Collagen molecules are crucial extracellular players in animal tissue development and in functions ranging from ultrafiltration to organism locomotion. Among the 28 types of collagen found in human, type IV collagen stands out as a primordial type found in all species of the animal kingdom. Collagen IV forms smart scaffolds for basement membranes, sheet-like acellular structures that isolate, coordinate, and direct cells during morphogenesis. Collagen IV is also involved in multiple functions in developed tissues. As part of the basement membrane, collagen IV scaffolds provide mechanical strength, spatially tether extracellular macromolecules and directly signal to cells via receptor binding sites. Proper assembly and structure of the scaffolds are critical for development and function of multiple types of basement membranes. Within last 5 years it was established that Cl- concentration is a key factor for initiating collagen IV scaffold assembly. The biological role of Cl- in multiple physiological processes and detailed mechanisms for its signaling and structural impacts are well established. Cl- gradients are generated across the plasma and intracellular organelle membranes. As collagen IV molecules are secreted outside the cell, they experience a switch from low to high Cl- concentration. This transition works as a trigger for collagen IV scaffold assembly. Within the scaffold, collagen IV remains to be a Cl- sensor as its structural integrity continues to depend on Cl- concentration. Here, we review recent findings and set future directions for studies on the role of Cl- in type IV collagen assembly, function, and disease.


Subject(s)
Collagen Type IV , Animals , Basement Membrane , Humans , Morphogenesis
9.
J Biol Chem ; 294(20): 7968-7981, 2019 05 17.
Article in English | MEDLINE | ID: mdl-30923125

ABSTRACT

Collagen IV scaffold is a principal component of the basement membrane (BM), a specialized extracellular matrix that is essential for animal multicellularity and tissue evolution. Scaffold assembly begins with the trimerization of α-chains into protomers inside the cell, which then are secreted and undergo oligomerization outside the cell. For the ubiquitous scaffold composed of α1- and α2-chains, both intracellular and extracellular stages are mediated by the noncollagenous domain (NC1). The association of protomers is chloride-dependent, whereby chloride ions induce interactions of the protomers' trimeric NC1 domains leading to NC1 hexamer formation. Here, we investigated the mechanisms, kinetics, and functionality of the chloride ion-mediated protomer assembly by using a single-chain technology to produce a stable NC1 trimer comprising α1, α2, and α1 NC1 monomers. We observed that in the presence of chloride, the single-chain NC1-trimer self-assembles into a hexamer, for which the crystal structure was determined. We discovered that a chloride ring, comprising 12 ions, induces the assembly of and stabilizes the NC1 hexamer. Furthermore, we found that the chloride ring is evolutionarily conserved across all animals, first appearing in cnidarians. These findings reveal a fundamental role for the chloride ring in the assembly of collagen IV scaffolds of BMs, a critical event enabling tissue evolution and development. Moreover, the single-chain technology is foundational for generating trimeric NC1 domains of other α-chain compositions to investigate the α121, α345, and α565 collagen IV scaffolds and to develop therapies for managing Alport syndrome, Goodpasture's disease, and cancerous tumor growth.


Subject(s)
Collagen Type IV/chemistry , Models, Molecular , Crystallography, X-Ray , Humans , Protein Structure, Quaternary , Protein Structure, Tertiary
10.
J Biol Chem ; 293(16): 5987-5999, 2018 04 20.
Article in English | MEDLINE | ID: mdl-29491144

ABSTRACT

Type IV collagen is a major component of the basement membrane and interacts with numerous other basement membrane proteins. Many of these interactions are poorly characterized. Type IV collagen is abundantly post-translationally modified with 3-hydroxyproline (3-Hyp), but 3-Hyp's biochemical role in type IV collagen's interactions with other proteins is not well established. In this work, we present binding data consistent with a major role of 3-Hyp in interactions of collagen IV with glycoprotein VI and nidogens 1 and 2. The increased binding interaction between type IV collagen without 3-Hyp and glycoprotein VI has been the subject of some controversy, which we sought to explore, whereas the lack of binding of nidogens to type IV collagen without 3-Hyp is novel. Using tandem MS, we show that the putative glycoprotein VI-binding site is 3-Hyp-modified in WT PFHR-9 type IV collagen, but not in PFHR-9 cells in which prolyl-3-hydroxylase 2 (P3H2) has been knocked out (KO). Moreover, we observed altered 3-Hyp occupancy across many other sites. Using amino acid analysis of type IV collagen from the WT and P3H2 KO cell lines, we confirm that P3H2 is the major, but not the only 3-Hyp-modifying enzyme of type IV collagen. These findings underscore the importance of post-translational modifications of type IV collagen for interactions with other proteins.


Subject(s)
Collagen Type IV/metabolism , Hydroxyproline/metabolism , Membrane Glycoproteins/metabolism , Platelet Membrane Glycoproteins/metabolism , Protein Interaction Maps , Animals , Calcium-Binding Proteins , Cell Adhesion Molecules , Cell Line , Mice , Protein Processing, Post-Translational
11.
Proc Natl Acad Sci U S A ; 111(1): 161-6, 2014 Jan 07.
Article in English | MEDLINE | ID: mdl-24368846

ABSTRACT

Collagens constitute nearly 30% of all proteins in our body. Type IV collagen is a major and crucial component of basement membranes. Collagen chains undergo several posttranslational modifications that are indispensable for proper collagen function. One of these modifications, prolyl 3-hydroxylation, is accomplished by a family of prolyl 3-hydroxylases (P3H1, P3H2, and P3H3). The present study shows that P3H2-null mice are embryonic-lethal by embryonic day 8.5. The mechanism of the unexpectedly early lethality involves the interaction of non-3-hydroxylated embryonic type IV collagen with the maternal platelet-specific glycoprotein VI (GPVI). This interaction results in maternal platelet aggregation, thrombosis of the maternal blood, and death of the embryo. The phenotype is completely rescued by producing double KOs of P3H2 and GPVI. Double nulls are viable and fertile. Under normal conditions, subendothelial collagens bear the GPVI-binding sites that initiate platelet aggregation upon blood exposure during injuries. In type IV collagen, these sites are normally 3-hydroxylated. Thus, prolyl 3-hydroxylation of type IV collagen has an important function preventing maternal platelet aggregation in response to the early developing embryo. A unique link between blood coagulation and the ECM is established. The newly described mechanism may elucidate some unexplained fetal losses in humans, where thrombosis is often observed at the maternal/fetal interface. Moreover, epigenetic silencing of P3H2 in breast cancers implies that the interaction between GPVI and non-3-hydroxylated type IV collagen might also play a role in the progression of malignant tumors and metastasis.


Subject(s)
Collagen Type IV/metabolism , Procollagen-Proline Dioxygenase/metabolism , Amino Acid Sequence , Animals , Blood Coagulation , Cattle , Collagen Type IV/chemistry , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , Humans , Hydroxylation , Mass Spectrometry , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Sequence Data , Phenotype , Platelet Aggregation , Procollagen-Proline Dioxygenase/chemistry , Protein Structure, Tertiary , Thrombosis , Time Factors
12.
J Biol Chem ; 288(34): 24742-52, 2013 Aug 23.
Article in English | MEDLINE | ID: mdl-23861401

ABSTRACT

Type I collagen extracted from tendon, skin, and bone of wild type and prolyl 3-hydroxylase 1 (P3H1) null mice shows distinct patterns of 3-hydroxylation and glycosylation of hydroxylysine residues. The A1 site (Pro-986) in the α1-chain of type I collagen is almost completely 3-hydroxylated in every tissue of the wild type mice. In contrast, no 3-hydroxylation of this proline residue was found in P3H1 null mice. Partial 3-hydroxylation of the A3 site (Pro-707) was present in tendon and bone, but absent in skin in both α-chains of the wild type animals. Type I collagen extracted from bone of P3H1 null mice shows a large reduction in 3-hydroxylation of the A3 site in both α-chains, whereas type I collagen extracted from tendon of P3H1 null mice shows little difference as compared with wild type. These results demonstrate that the A1 site in type I collagen is exclusively 3-hydroxylated by P3H1, and presumably, this enzyme is required for the 3-hydroxylation of the A3 site of both α-chains in bone but not in tendon. The increase in glycosylation of hydroxylysine in P3H1 null mice in bone was found to be due to an increased occupancy of normally glycosylated sites. Despite the severe disorganization of collagen fibrils in adult tissues, the D-period of the fibrils is unchanged. Tendon fibrils of newborn P3H1 null mice are well organized with only a slight increase in diameter. The absence of 3-hydroxyproline and/or the increased glycosylation of hydroxylysine in type I collagen disturbs the lateral growth of the fibrils.


Subject(s)
Collagen Type I/metabolism , Procollagen-Proline Dioxygenase/metabolism , Protein Processing, Post-Translational/physiology , Animals , Collagen Type I/genetics , Hydroxylation/physiology , Mice , Mice, Mutant Strains , Organ Specificity/physiology , Procollagen-Proline Dioxygenase/genetics , Proline/genetics , Proline/metabolism
13.
Matrix Biol ; 32(1): 39-44, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23186870

ABSTRACT

Prolyl 3-hydroxylase1 (P3H1) is a collagen modifying enzyme which hydroxylates certain prolines in the Xaa position of conventional GlyXaaYaa triple helical sequence. Recent investigations have revealed that mutations in the LEPRE1 (gene encoding for P3H1) cause severe osteogenesis imperfecta (OI) in humans. Similarly LEPRE1 knockout mice display an OI-like phenotype. Significant hearing loss is a common problem for people with osteogenesis imperfecta. Here we report that hearing of the P3H1 null mice is substantially affected. Auditory brainstem responses (ABRs) of the P3H1 null mice show an average increase of 20-30 dB in auditory thresholds. Three dimensional reconstructions of the mutant middle ear bones by Micro-scale X-ray computed tomography (Micro-CT) demonstrate abnormal morphology of the incudostapedial and incudomalleal joints. We establish the LEPRE1 knockout mouse as a valuable model system to investigate the mechanism of hearing loss in recessive OI.


Subject(s)
Ear Ossicles/abnormalities , Hearing Loss/genetics , Joints/abnormalities , Membrane Glycoproteins/genetics , Osteogenesis Imperfecta/genetics , Procollagen-Proline Dioxygenase/deficiency , Proteoglycans/genetics , Animals , Auditory Threshold , Evoked Potentials, Auditory, Brain Stem/physiology , Genes, Recessive/genetics , Hearing Loss/enzymology , Mice , Mice, Knockout , Osteogenesis Imperfecta/enzymology , X-Ray Microtomography
14.
J Biol Chem ; 287(26): 22253-65, 2012 Jun 22.
Article in English | MEDLINE | ID: mdl-22556420

ABSTRACT

The rate-limiting step of folding of the collagen triple helix is catalyzed by cyclophilin B (CypB). The G6R mutation in cyclophilin B found in the American Quarter Horse leads to autosomal recessive hyperelastosis cutis, also known as hereditary equine regional dermal asthenia. The mutant protein shows small structural changes in the region of the mutation at the side opposite the catalytic domain of CypB. The peptidylprolyl cis-trans isomerase activity of the mutant CypB is normal when analyzed in vitro. However, the biosynthesis of type I collagen in affected horse fibroblasts shows a delay in folding and secretion and a decrease in hydroxylysine and glucosyl-galactosyl hydroxylysine. This leads to changes in the structure of collagen fibrils in tendon, similar to those observed in P3H1 null mice. In contrast to cyclophilin B null mice, where little 3-hydroxylation was found in type I collagen, 3-hydroxylation of type I collagen in affected horses is normal. The mutation disrupts the interaction of cyclophilin B with the P-domain of calreticulin, with lysyl hydroxylase 1, and probably other proteins, such as the formation of the P3H1·CypB·cartilage-associated protein complex, resulting in less effective catalysis of the rate-limiting step in collagen folding in the rough endoplasmic reticulum.


Subject(s)
Collagen/chemistry , Cyclophilins/genetics , Mutation , Peptidylprolyl Isomerase/chemistry , Skin Diseases/genetics , Skin Diseases/veterinary , cis-trans-Isomerases/metabolism , Animals , Asthenia , Circular Dichroism , Endoplasmic Reticulum, Rough/metabolism , Horses , Kinetics , Mice , Mice, Transgenic , Molecular Chaperones/metabolism , Protein Binding , Protein Folding , Protein Structure, Tertiary , Surface Plasmon Resonance
15.
J Biol Chem ; 285(22): 17253-62, 2010 May 28.
Article in English | MEDLINE | ID: mdl-20363744

ABSTRACT

Osteogenesis imperfecta (OI) is a skeletal disorder primarily caused by mutations in the type I collagen genes. However, recent investigations have revealed that mutations in the genes encoding for cartilage-associated protein (CRTAP) or prolyl 3-hydroxylase 1 (P3H1) can cause a severe, recessive form of OI. These reports show minimal 3-hydroxylation of key proline residues in type I collagen as a result of CRTAP or P3H1 deficiency and demonstrate the importance of P3H1 and CRTAP to bone structure and development. P3H1 and CRTAP have previously been shown to form a stable complex with cyclophilin B, and P3H1 was shown to catalyze the 3-hydroxylation of specific proline residues in procollagen I in vitro. Here we describe a mouse model in which the P3H1 gene has been inactivated. Our data demonstrate abnormalities in collagen fibril ultrastructure in tendons from P3H1 null mice by electron microscopy. Differences are also seen in skin architecture, as well as in developing limbs by histology. Additionally bone mass and strength were significantly lower in the P3H1 mice as compared with wild-type littermates. Altogether these investigations demonstrate disturbances of collagen fiber architecture in tissues rich in fibrillar collagen, including bone, tendon, and skin. This model system presents a good opportunity to study the underlying mechanisms of recessive OI and to better understand its effects in humans.


Subject(s)
Bone and Bones/metabolism , Collagen/chemistry , Procollagen-Proline Dioxygenase/genetics , Procollagen-Proline Dioxygenase/physiology , Skin/metabolism , Tendons/metabolism , Animals , Bone and Bones/embryology , Bone and Bones/ultrastructure , Extracellular Matrix/metabolism , Gene Expression Regulation, Developmental , Hydrolysis , Mice , Mice, Knockout , Mice, Transgenic , Microscopy, Electron/methods , Osteogenesis Imperfecta/metabolism , Protein Processing, Post-Translational , Skin/embryology , Skin/ultrastructure , Tendons/embryology , Tendons/ultrastructure
16.
Cell ; 124(3): 485-93, 2006 Feb 10.
Article in English | MEDLINE | ID: mdl-16469696

ABSTRACT

Dengue virus (DENV) is a significant human pathogen that causes millions of infections and results in about 24,000 deaths each year. Dendritic cell-specific ICAM3 grabbing nonintegrin (DC-SIGN), abundant in immature dendritic cells, was previously reported as being an ancillary receptor interacting with the surface of DENV. The structure of DENV in complex with the carbohydrate recognition domain (CRD) of DC-SIGN was determined by cryo-electron microscopy at 25 A resolution. One CRD monomer was found to bind to two glycosylation sites at Asn67 of two neighboring glycoproteins in each icosahedral asymmetric unit, leaving the third Asn67 residue vacant. The vacancy at the third Asn67 site is a result of the nonequivalence of the glycoprotein environments, leaving space for the primary receptor binding to domain III of E. The use of carbohydrate moieties for receptor binding sites suggests a mechanism for avoiding immune surveillance.


Subject(s)
Cell Adhesion Molecules/chemistry , Dengue Virus/chemistry , Dengue Virus/ultrastructure , Lectins, C-Type/chemistry , Receptors, Cell Surface/chemistry , Binding Sites , Carbohydrates/chemistry , Cryoelectron Microscopy , Dendritic Cells/virology , Dengue Virus/pathogenicity , Humans , Image Processing, Computer-Assisted , Models, Molecular , Multiprotein Complexes , Protein Structure, Tertiary , Receptors, Virus/chemistry , Recombinant Proteins/chemistry
17.
J Biol Chem ; 279(50): 52016-23, 2004 Dec 10.
Article in English | MEDLINE | ID: mdl-15466471

ABSTRACT

The nematocyst is a unique extrusive organelle involved in the defense and capture of prey in cnidarians. Minicollagens and the glycoprotein NOWA are major components of the nematocyst capsule wall, which resists osmotic pressure of 15 MPa. Here we present the recombinant expression of NOWA, which spontaneously assembles to globular macromolecular particles that are sensitive to reduction as the native wall structure. Ultra-structural analysis showed that the Hydra nematocyst wall is composed of several layers of globular particles, which are interconnected via radiating rodlike protrusions. Evidence is presented that native wall particles contain NOWA and minicollagen, supposed to be linked via disulfide bonds between their homologous cysteine-rich domains. Our data suggest a continuous suprastructure of the nematocyst wall, assembled from wall proteins that share a common oligomerization motif.


Subject(s)
Hydra/metabolism , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/metabolism , Animals , Base Sequence , Biopolymers/chemistry , Biopolymers/genetics , Biopolymers/metabolism , Collagen/chemistry , Collagen/metabolism , DNA/genetics , Disulfides/chemistry , Hydra/genetics , Hydra/ultrastructure , Membrane Glycoproteins/genetics , Microscopy, Electron , Microscopy, Electron, Scanning , Organelles/metabolism , Organelles/ultrastructure , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
18.
FEBS Lett ; 569(1-3): 112-6, 2004 Jul 02.
Article in English | MEDLINE | ID: mdl-15225618

ABSTRACT

A high-precision solution structure of the C-terminal minicollagen cysteine rich domain of Hydra has been determined using modern heteronuclear and weak alignment NMR techniques at natural isotope abundance. The domain consists of only 24 amino acids, six of which are prolines and six are cysteines bonded in disulfide bridges that constrain the structure into a new fold. The redox equilibrium of the structure has been characterized from a titration with glutathione. No local native structures are detectable in the reduced form. Thus, oxidation and folding are tightly coupled.


Subject(s)
Collagen/chemistry , Cysteine/analysis , Hydra/chemistry , Amino Acid Sequence , Animals , Disulfides/analysis , Glutathione/analysis , Glutathione Disulfide/analysis , Hydrogen Bonding , Magnetic Resonance Spectroscopy/methods , Molecular Sequence Data
19.
J Biol Chem ; 279(29): 30395-401, 2004 Jul 16.
Article in English | MEDLINE | ID: mdl-15123641

ABSTRACT

The minicollagens found in the nematocysts of Hydra constitute a family of invertebrate collagens with unusual properties. They share a common modular architecture with a central collagen sequence ranging from 14 to 16 Gly-X-Y repeats flanked by polyproline/hydroxyproline stretches and short terminal domains that show a conserved cysteine pattern (CXXXCXXXCXXX-CXXXCC). The minicollagen cysteine-rich domains are believed to function in a switch of the disulfide connectivity from intra- to intermolecular bonds during maturation of the capsule wall. The solution structure of the C-terminal fragment including a minicollagen cysteine-rich domain of minicollagen-1 was determined in two independent groups by 1H NMR. The corresponding peptide comprising the last 24 residues of the molecule was produced synthetically and refolded by oxidation under low protein concentrations. Both presented structures are identical in their fold and disulfide connections (Cys2-Cys18, Cys6-Cys14, and Cys10-Cys19) revealing a robust structural motif that is supposed to serve as the polymerization module of the nematocyst capsule.


Subject(s)
Collagen/chemistry , Cysteine/chemistry , Amino Acid Motifs , Amino Acid Sequence , Animals , Binding Sites , Cystine/chemistry , Disulfides , Glycine/chemistry , Hydra , Kinetics , Magnetic Resonance Spectroscopy , Mass Spectrometry , Models, Molecular , Molecular Sequence Data , Oxygen/metabolism , Peptides/chemistry , Protein Conformation , Protein Folding , Protein Isoforms , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Time Factors , Ultracentrifugation
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