Biomedical subjects

Karl E Kadler

Publications and source records attributed to Karl E Kadler.

14 recordsLinked to original sources

PubMed2006

The 10+4 microfibril structure of thin cartilage fibrils.

Determining the structure of cartilage collagen fibrils will provide insights into how mutations in collagen genes affect cartilage formation during skeletal morphogenesis and understanding the mechanism of fibril growth. The fibrils are indeterminate in size, heteropolymeric, and highly cross-linked, which make them refractory to analysis by conventional high-resolution structure determination techniques. Electron microscopy has been limited to making simple measurements of fibril diameter and immunolocalizing certain molecules at the fibril surface. Consequently, structural information on the fibrils is limited. In this study we have used scanning transmission electron microscopic mass mapping, analysis of axial stain exclusion pattern, and r-weighted back-projection techniques to determine the intermediate resolution (to approximately 4 nm) structure of thin collagen fibrils from embryonic cartilage. The analyses show that the fibrils are constructed from a 10+4 microfibrillar arrangement in which a core of four microfibrils is surrounded by a ring of 10 microfibrils. Accurate mass measurements predict that each microfibril contains five collagen molecules in cross-section. Based on the proportion of collagen II, IX, and XI in the fibrils, the fibril core comprises two microfibrils each of collagen II and collagen XI. Single molecules of collagen IX presumably occur at the fibril surface between the extended N-terminal domains of collagen XI. The 10+4 microfibril structure explains the mechanism of diameter limitation in the narrow fibrils and the absence of narrow collagen fibrils in cartilage lacking collagen XI.

Animals↗

PubMed2006

Actin filaments are required for fibripositor-mediated collagen fibril alignment in tendon.

Cells in tendon deposit parallel arrays of collagen fibrils to form a functional tissue, but how this is achieved is unknown. The cellular mechanism is thought to involve the formation of intracellular collagen fibrils within Golgi to plasma membrane carriers. This is facilitated by the intracellular processing of procollagen to collagen by members of the tolloid and ADAMTS families of enzymes. The carriers subsequently connect to the extracellular matrix via finger-like projections of the plasma membrane, known as fibripositors. In this study we have shown, using three-dimensional electron microscopy, the alignment of fibripositors with intracellular fibrils as well as an orientated cable of actin filaments lining the cytosolic face of a fibripositor. To demonstrate a specific role for the cytoskeleton in coordinating extracellular matrix assembly, cytochalasin was used to disassemble actin filaments and nocodazole or colchicine were used to disrupt microtubules. Microtubule disruption delayed procollagen transport through the secretory pathway, but fibripositor numbers were unaffected. Actin filament disassembly resulted in rapid loss of fibripositors and a subsequent disappearance of intracellular fibrils. Procollagen secretion or processing was not affected by cytochalasin treatment, but the parallelism of extracellular collagen fibrils was altered. In this case a significant proportion of collagen fibrils were found to no longer be orientated with the long axis of the tendon. The results suggest an important role for the actin cytoskeleton in the alignment and organization of the collagenous extracellular matrix in embryonic tendon.

Actins↗

PubMed2006

A complete domain structure of Drosophila tolloid is required for cleavage of short gastrulation.

Drosophila tolloid (TLD) is a member of a family of proteinases that play important roles in development and includes mammalian tolloid (mTLD) and bone morphogenetic protein (BMP)-1. TLD accentuates the activity of decapentaplegic (DPP), a transforming growth factor beta superfamily growth factor, by cleaving its antagonist Short gastrulation (Sog). Similarly, the activity of BMP-2/4 (vertebrate homologues of DPP) is augmented by cleavage of chordin. However, whereas TLD is an effective Sogase, mTLD is a poor chordinase and is functionally replaced by its smaller splice variant BMP-1, which lacks the most C-terminal epidermal growth factor (EGF)-like and CUB domains of mTLD. Moreover, the minimal chordinase activity resides in the N-terminal half of BMP-1. This study showed that the proteolytic activity of TLD is considerably enhanced by Ca2+ and tested the hypothesis that the Sogase activity of TLD resides in the N-terminal half of the proteinase. Unexpectedly, it was found that TLD lacking the CUB4 and CUB5 domains and/or the EGF-like domains was unable to cleave Sog. Loss of function mutations have been reported in the tld gene that result in amino acid substitutions at E835K (in CUB4), S915L (in CUB5), and N760I (in EGF2) in TLD. The CUB mutants were found to be ineffective Sogases, but the activity of the EGF2 mutant was unchanged. The results show that substrate recognition and cleavage by Drosophila tolloid and mTLD are different despite their identical domain structure and homologous functions in patterning. The result that the N760I mutant has full Sogase activity suggests that novel substrates for TLD exist.

Amino Acid Sequence↗

PubMed2005

Identification of the minimal domain structure of bone morphogenetic protein-1 (BMP-1) for chordinase activity: chordinase activity is not enhanced by procollagen C-proteinase enhancer-1 (PCPE-1).

Bone morphogenetic protein 1 (BMP-1), which is a tolloid member of the astacin-like family of zinc metalloproteinases, is a highly effective procollagen C-proteinase (PCP) and chordinase. On the other hand, mammalian tolloid like-2 (mTLL-2) does not cleave chordin or procollagen; procollagen is cleaved by mTLL-2 in the presence of high levels of procollagen C-proteinase enhancer-1 (PCPE-1), for reasons that are unknown. We used these differences in activity between BMP-1 and mTLL-2 to narrow in on the domains in BMP-1 that specify PCP and chordinase activity. Using a domain swap approach, we showed that: 1) the metalloproteinase and CUB2 domains of BMP-1 are absolutely required for PCP activity; swaps with either of the corresponding domains in BMP-1 and mTLL-2 did not result in procollagen cleavage and 2) the proteinase domain of mTLL-2 can cleave chordin if coupled to the CUB1 domain of BMP-1. Therefore, the minimal structure for chordinase activity comprises a metalloproteinase domain (either from BMP-1 or from mTLL-2) and the CUB1 domain of BMP-1 (the CUB1 domain of mTLL-2 cannot substitute for the CUB1 domain of BMP-1). We showed that the minimal procollagen C-proteinase (BMP-1 lacking the EGF and CUB3 domain) was enhanced by PCPE-1 but not as well as BMP-1 retaining the CUB3 domain. Further studies showed that PCPE-1 had no effect on the ability of BMP-1 to cleave chordin. The data support a previously suggested mechanism of PCPE-1 whereby PCPE-1 interacts with procollagen, but in addition, the CUB3 domain of BMP-1 appears to augment the interaction.

Blotting, Western↗

PubMed2005

Procollagen trafficking, processing and fibrillogenesis.

Collagen fibrils in the extracellular matrix allow connective tissues such as tendon, skin and bone to withstand tensile forces. The fibrils are indeterminate in length, insoluble and form elaborate three-dimensional arrays that extend over numerous cell lengths. Studies of the molecular basis of collagen fibrillogenesis have provided insight into the trafficking of procollagen (the precursor of collagen) through the cellular secretory pathway, the conversion of procollagen to collagen by the procollagen metalloproteinases, and the directional deposition of fibrils involving the plasma membrane and late secretory pathway. Fibril-associated molecules are targeted to the surface of collagen fibrils, and these molecules play an important role in regulating the diameter and interactions between the fibrils.

Animals↗

PubMed2005

The precision of lateral size control in the assembly of corneal collagen fibrils.

Collagen fibrils in the corneal stroma have been recognised to have a high degree of uniformity of diameter and spatial arrangement compared with those in other mature connective tissues. The precision of this lateral size control has been determined in this study by mass per unit length measurements on fibrils isolated from adult bovine corneal stroma. At the molecular level, however, there are substantial variations in lateral size, both between fibrils and along individual fibrils. The mean mass per unit length was measured to be 304 kDa nm(-1), equivalent to 347 collagen molecules in transverse section and had a standard deviation of 8.3%. The variation of lateral size along individual fibrils was measured as a mass slope over approximately 7 microm lengths (100 D-periods) and had a mean mass slope equivalent to 0.56 molecules per D-period. Smoothly tapered tips of length approximately 7 microm were also observed with a mass slope of about approximately three molecules per D-period. The frequency of these tips was used to estimate a mean fibril length of approximately 940 microm in the sample tissue. Observations of molecular polarity within the fibril shafts and tips were used to consider possible models of fibril assembly.

Animals↗

PubMed2004

Analysis of collagen fibril diameter distribution in connective tissues using small-angle X-ray scattering.

Analysis of the diameters of collagen fibrils provides insight into the structure and physical processes occurring in the tissue. This paper describes a method for analyzing the frequency distribution of the diameters of collagen fibrils from small-angle X-ray scattering (SAXS) patterns. Frequency values of fibril diameters were input into a mathematical model of the form factor to calculate the equatorial intensity which best fits the experimentally derived data from SAXS patterns. A minimization algorithm utilizing simulated annealing (SA) was used in the fitting procedure. The SA algorithm allowed for random sampling of the frequency values, and was run iteratively to build up an optimized frequency distribution of fibril diameters. Results were obtained for collagen samples from sheep spine ligaments. The mean fibril diameter value obtained from this data-fitting method was 73 nm+/-20 nm (S.D.). From scanning transmission electron microscopy, the mean diameter was found to be 69 nm+/-14 nm (S.D.). The good agreement between the two methods demonstrates the reliability of the SAXS method for the tissue examined. The non-destructive nature of this technique, as well as its statistical robusticity and capacity for large sampling, means that this method is both quick and effective.

Algorithms↗

PubMed2004

Deletion of epidermal growth factor-like domains converts mammalian tolloid into a chordinase and effective procollagen C-proteinase.

Bone morphogenetic protein (BMP)-1 and mammalian tolloid (mTld) are Ca(2+)-dependent metalloproteinases that result from alternative splicing of the bmp1 gene. They have different proteinase activities, e.g. BMP-1 effectively cleaves procollagen (an extracellular matrix protein) and chordin (a BMP antagonist), whereas mTld is a poor procollagen proteinase and will not cleave chordin in the absence of twisted gastrulation. This is perplexing because mTld (being the longer variant) might be expected to cleave all substrates cleaved by BMP-1. Studies have shown that the minimal structure for procollagen proteinase activity is proteinase-CUB1-CUB2 (BMP-1DeltaEC3) and therefore lacking the epidermal growth factor (EGF)-like domain thought to account for the Ca(2+) dependence of BMP-1. In this study we generated three deletion mutants of mTld that lacked either one or both EGF-like domains (referred to as "mTld-DeltaEGF"). The mutated proteins were poorly but sufficiently secreted from 293-EBNA cells for in vitro assays of procollagen and chordin cleavage. Most surprisingly, the mTld-DeltaEGF mutants required Ca(2+) for proteolytic activity, thereby showing that the EGF-like domains do not account for the Ca(2+) dependence of BMP-1/mTld. Moreover, the mTld-DeltaEGFs are effective procollagen proteinases and cleave chordin. Furthermore, BMP-1DeltaEC3 cleaves chordin and requires Ca(2+) for activity. Studies using nondenaturing gels showed that mTld molecules lacking EGF-like domains have a loose conformation such that in the presence of Ca(2+) binding sites for chordin and procollagen on the "BMP-1-part" of the molecule are exposed. We propose that the EGF-like domains could hold CUB4/5 domains in locations that exclude substrates cleavable by BMP-1.

Amino Acid Sequence↗

PubMed2004

Coalignment of plasma membrane channels and protrusions (fibripositors) specifies the parallelism of tendon.

The functional properties of tendon require an extracellular matrix (ECM) rich in elongated collagen fibrils in parallel register. We sought to understand how embryonic fibroblasts elaborate this exquisite arrangement of fibrils. We show that procollagen processing and collagen fibrillogenesis are initiated in Golgi to plasma membrane carriers (GPCs). These carriers and their cargo of 28-nm-diam fibrils are targeted to previously unidentified plasma membrane (PM) protrusions (here designated "fibripositors") that are parallel to the tendon axis and project into parallel channels between cells. The base of the fibripositor lumen (buried several microns within the cell) is a nucleation site of collagen fibrillogenesis. The tip of the fibripositor is the site of fibril deposition to the ECM. Fibripositors are absent at postnatal stages when fibrils increase in diameter by accretion of extracellular collagen, thereby maintaining parallelism of the tendon. Thus, we show that the parallelism of tendon is determined by the late secretory pathway and interaction of adjacent PMs to form extracellular channels.

Animals↗

PubMed2004

Age-related changes on the surface of vitreous collagen fibrils.

PURPOSE: To determine whether aging vitreous collagen fibrils undergo ultrastructural changes that might underlie vitreous liquefaction and posterior vitreous detachment. METHODS: Vitreous collagen fibrils from 21 human subjects (age range, 3-89 years) and from bovine eyes were isolated on electron microscopy grids. Cupromeronic blue labeling in the presence of 0.3 M MgCl(2) and immunogold labeling for collagen types II and IX were analyzed by transmission electron microscopy. RESULTS: Aging was associated with marked changes on the surface of human vitreous collagen fibrils, including an exponential loss of type IX collagen along with its chondroitin sulfate side-chains (half-life, 11 years) and a fourfold increase in the exposure of type II collagen. CONCLUSIONS: Despite being a minor component of vitreous collagen fibrils, type IX collagen, probably by virtue of its chondroitin sulfate side-chains, shields type II collagen from exposure on the fibril surface. With aging, this shielding diminishes, resulting in the surface exposure of "sticky" type II collagen and thus predisposing the vitreous collagen fibrils to fusion. These changes could underlie vitreous liquefaction and weakening of vitreoretinal adhesion.

Adolescent↗

PubMed2003

Bone morphogenetic protein-1 (BMP-1). Identification of the minimal domain structure for procollagen C-proteinase activity.

Bone morphogenetic protein-1 (BMP-1) is a shorter spliced variant of mammalian tolloid (mTld), both of which cleave the C-propeptides of type I procollagen during the synthesis of extracellular matrix collagen fibrils. The fact that BMP-1 and mTld both exhibit procollagen C-proteinase (PCP) activity and that BMP-1 is the smaller variant might indicate that BMP-1 comprises the minimal required sequences for PCP activity. BMP-1 comprises a metalloproteinase domain, three CUB domains, and an epidermal growth factor (EGF)-like domain, which is located between the second and third CUB (complement components C1r/C1s, the sea urchin protein Uegf, and BMP-1) domains. In this study we showed the following. 1) The CUB1 domain is required for secretion of the molecule. Domain swapping experiments, in which CUB1 and other CUB domains were interchanged, resulted in retention of the proteins by cells. Therefore, CUB1 and its location immediately adjacent to the metalloproteinase domain are essential for secretion of the protein. 2) Mutants lacking the EGF-like and CUB3 domains exhibited full C-proteinase activity. In contrast, mutants lacking the CUB2 domain were poor C-proteinases. 3) Further studies showed that Glu-483 on the beta4-beta5 loop of CUB2 is essential for C-proteinase activity of BMP-1. In conclusion, the study showed that the minimal domain structure for PCP activity is considerably shorter than expected and comprises the metalloproteinase domain and the CUB1 and CUB2 domains of BMP-1.

Blotting, Western↗

PubMed2003

Paired basic/Furin-like proprotein convertase cleavage of Pro-BMP-1 in the trans-Golgi network.

Bone morphogenetic protein (BMP)-1 is a zinc-dependent metalloproteinase that cleaves a variety of extracellular matrix substrates, including type I procollagen. Little is known about the site of action of BMP-1, although the extracellular matrix seems likely to be it. BMP-1 is synthesized with an N-terminal prodomain. The removal of the prodomain presumably activates the proteinase. In this study we show that the prodomain is cleaved in the trans-Golgi network (TGN) and by furin-like/paired basic proprotein convertases. Inhibitors of furin resulted in the secretion of pro-BMP-1, which could not cleave procollagen. Recombinant furin cleaved the prodomain from pro-BMP-1. Site-directed mutagenesis of the prodomain cleavage site (RSRR) to RSAA resulted in efficient secretion of pro-BMP-1. Therefore, prodomain cleavage was not required for secretion. Using peptide N-glycosidase and neuraminidase digestion to determine the post-translational status of pro-BMP-1 during its conversion to BMP-1, we showed that BMP-1 first appears in the TGN during sialylation of the molecule. Furthermore, immunofluorescence studies using an antibody to the nascent N terminus of BMP-1 showed localization to the TGN and plasma membrane. The observation that BMP-1 occurs inside the cell raises the possibility that BMP-1 might begin to cleave its substrates prior to secretion to the extracellular matrix.

Amidohydrolases↗

PubMed2002

Post-translational modification of bone morphogenetic protein-1 is required for secretion and stability of the protein.

Bone morphogenetic protein (BMP)-1 is a glycosylated metalloproteinase that is fundamental to the synthesis of a normal extracellular matrix because it cleaves type I procollagen, as well as other precursor proteins. Sequence analysis suggests that BMP-1 has six potential N-linked glycosylation sites (i.e. NXS/T) namely: Asn(91) (prodomain), Asn(142) (metalloproteinase domain), Asn(332) and Asn(363) (CUB1 domain), Asn(599) (CUB3 domain), and Asn(726) in the C-terminal-specific domain. In this study we showed that all these sites are N-glycosylated with complex-type oligosaccharides containing sialic acid, except Asn(726) presumably because proline occurs immediately C-terminal of threonine in the consensus sequence. Recombinant BMP-1 molecules lacking all glycosylation sites or the three CUB-specific sites were not secreted. BMP-1 lacking CUB glycosylation was translocated to the proteasome for degradation. BMP-1 molecules lacking individual glycosylation sites were efficiently secreted and exhibited full procollagen C-proteinase activity, but N332Q and N599Q exhibited a slower rate of cleavage. BMP-1 molecules lacking any one of the CUB-specific glycosylation sites were sensitive to thermal denaturation. The study showed that the glycosylation sites in the CUB domains of BMP-1 are important for secretion and stability of the molecule.

Amino Acid Substitution↗

PubMed2002

Proteinases of the bone morphogenetic protein-1 family convert procollagen VII to mature anchoring fibril collagen.

Collagen VII is the major structural component of the anchoring fibrils at the dermal-epidermal junction in the skin. It is secreted by keratinocytes as a precursor, procollagen VII, and processed into mature collagen during polymerization of the anchoring fibrils. We show that bone morphogenetic protein-1 (BMP-1), which exhibits procollagen C-proteinase activity, cleaves the C-terminal propeptide from human procollagen VII. The cleavage occurs at the BMP-1 consensus cleavage site SYAA/DTAG within the NC-2 domain. Mammalian tolloid-like (mTLL)-1 and -2, two other proteases of the astacin enzyme family, were able to process procollagen VII at the same site in vitro. Immunohistochemical and genetic evidence supported the involvement of these enzymes in cleaving type VII procollagen in vivo. Both BMP-1 and mTLL-1 are expressed in the skin and in cultured cutaneous cells. A naturally occurring deletion in the human COL7A1 gene, 8523del14, which is associated with dystrophic epidermolysis bullosa and eliminates the BMP-1 consensus sequence, abolished processing of procollagen VII, and in mutant skin procollagen VII accumulated at the dermal-epidermal junction. On the other hand, deficiency of BMP-1 in the skin of knockout mouse embryos did not prevent processing of procollagen VII to mature collagen, suggesting that mTLL-1 and/or mTLL-2 can substitute for BMP-1 in the processing of procollagen VII in situ.

Animals↗