Collagen polymorphism: its origins in the amino acid sequence.

The polymorphic forms of ordered collagen aggregation in vitro and in vivo are reviewed. The axially projected structures of a class of fibrils known as fibrous long spacing (FLS) collagen are solved using simulated positively stained banding patterns based on the amino acid sequence. This method is also used to solve the axial projection of a 670 Å (D) periodic structure with a symmetrical banding pattern (DPS) re-precipitated from skin collagen. The relation between the obliquely striated and 110 Å periodic forms of collagen is discussed. The specificity for the formation of FLS, DPS and segment long spacing (SLS) collagen is shown to be in the distributions of various amino acids in the sequence. Different residues are important for each type of structure, their importance being dependent on the chemical conditions and the presence of other macromolecules. The interaction of collagen fibrils with proteoglycans in vivo is discussed in terms of the amino acid sequence. Also the factors which affect collagen morphology in the presence of mucopolysaccharides and proteoglycans in vitro and in vivo are discussed. Some insight is gamed into the principles which govern the self-assembly of molecules into ordered fibrous aggregates.     

New Fibrous Long Spacing Form of Collagen

PRECIPITATION of collagen from solution in the presence of chondroitin sulphates (or certain other glycosaminoglycans), followed by dialysis against water, leads to the formation of the fibrous long spacing (FLS) polymorphic form of collagen. Electron microscopy shows this to consist of banded fibrils with a periodicity in the range 1200–2400 Å and a symmetric intraperiod structure, in contrast to the 600–700 Å periodicity and polarized fine structure characteristic of native collagen fibrils. FLS was originally reported by Highberger et al.^1–3 and has been observed in three principal modifications, FLS I, FLS II and FLS III, differing in periodicity and arrangement of dense bands^4–6. This letter reports a new form of FLS, denoted FLS IV.     

Purification and partial characterization of the type II procollagen synthesized by embryonic cartilage cells

Matrix-free cells were prepared from sternal cartilages of 17-day-old chick embryos, and procollagen synthesized and secreted by the cells was isolated by ion exchange chromatography on carboxymethyl cellulose and by gel filtration. The isolated protein was homogeneous by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and it appeared to consist of identical pro-α chains linked by interchain disulfide bonds. Amino acid analysis and cyanogen bromide peptide mapping of the purified procollagen demonstrated that it had structures similar to Type II collagen. The amino acid composition was also consistent with the conclusion that the peptide extensions on the pro-α chains of procollagen contained amino acid sequences not found in the collagen portion of the molecule. Segment-long-spacing aggregates were prepared from the procollagen, and aggregates demonstrated the same banding pattern as is found in segment-long-spacing aggregates prepared from Type II collagen. The segment-long-spacing aggregates from procollagen revealed, however, the presence of NH₂-terminal extensions of about 150 Å in length. In addition, the procollagen molecules contained irregularly shaped, large extension peptides at the COOH-terminal end of the molecule.     

The processing of procollagen in cultures of human and mouse fibroblasts.

Cultures of normal human and mouse fibroblasts convert procollagen to tropocollagen at varying rates. The conversion rate cannot be predicted from the species of origin of the fibroblast nor from the age of the donor tissue. Procollagen is converted to tropocollagen in both the extracellular space of the cell layer and in the culture medium. The collagen fibers of the cell layer are formed mostly from tropocollagen molecules generated in situ.     

Characterization of the tissue form of type V collagen from chick bone

Type V collagen was prepared from acetic acid extracts of lathyritic chick bone. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the extracted material demonstrated two collagenous bands of slower mobility than pepsin-extracted alpha 1(V) and alpha 2(V) chains. Cyanogen bromide peptide maps of these protein bands identified them as forms of alpha 1(V) and alpha 2(V). Segment long spacing (SLS) crystallite banding patterns of the acid-extracted Type V were identical within the triple-helical domain to the SLS banding patterns of pepsin-extracted Type V collagen, supporting the identification of this material. A globular domain at one end of the triple helix of the acid-extracted Type V was visualized by both rotary shadowing and negative staining of SLS crystallites. The molecular weights of the globular terminal peptides were 18,000 and 29,000, respectively, for alpha 1(V) and alpha 2(V), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after bacterial collagenase digestion of the isolated alpha chains. The results presented here indicate that fully processed Type V collagen in chick bone exists as a higher molecular weight form than that from pepsin extracts and retains a globular domain at one end of the triple helix. This is in contrast to the interstitial collagens in which only very small non-triple-helical domains (telopeptides) are retained in the fully processed molecules. In vitro aggregation studies demonstrated the intact fully processed form of Type V collagen forms uniform small-diameter fibrous structures. These results suggest that Type V collagen may be present in fibrous structures within tissues.     

The effects of dipalmitoyl phosphatidyl choline on the precipitation of native fibrils and segment-long-spacing aggregates from collagen solution

The effect of dipalmitoyl phosphatidyl choline (DPPC), the major phospholipid component of pulmonary surfactant, on the precipitation of collagen in the form of native fibrils and segment-long-spacing (SLS) aggregates was studied in vitro. The effects of DPPC on both phases of collagen fibrillogenesis were analyzed spectrophotometrically, and alterations in the morphology of precipitated fibrils and SLS aggregates were ascertained by transmission electron microscopy (TEM). Low concentrations of DPPC inhibited the growth phase of fibrillogenesis, while higher concentrations were required to inhibit nucleation. Both the meshwork density and mean width of precipitated fibrils were altered by DPPC, as was the size of SLS aggregates. Segment-long-spacing aggregates prepared from pepsin-treated collagen were inhibited to a greater degree than SLS aggregates prepared from untreated collagen, indicating that the pepsin-susceptible residues of the telopeptide extensions of tropocollagen molecules stabilize SLS aggregates against the effects of DPPC. Based on these results and the inhibition of the growth phase at lower concentrations than those which inhibited the nucleation phase of fibrillogenesis, it was concluded that the primary mechanism of DPPC inhibition is electrostatic interference between the positively charged phospholipid molecules and the net positive charge of collagen. It is proposed that pathological conditions involving the pulmonary epithelium may allow interaction between surfactant and collagen, which could further weaken the interstitial connective tissue.     

Human procollagen : I. An anionic tropocollagen precursor from skin fibroblasts in culture

Tropocollagen is derived from an extracellular precursor, procollagen. Conversion to tropocollagen is accomplished by one or more tissue pioteases dependent in vitro on the presence of serum in the culture medium. Twenty-four hour cultures in which serum has been excluded yield an apparently undegraded precursor, procollagen I. The latter is approximately twice the size of tropocollagen, possesses an acidic pl in contrast to the alkaline pl of tropocollagen, and shares secondary structural characteristics common to tropocollagen. Procollagen I exhibits a sharp thermal transition point at 39° with a ΔT of 2° indicating that the collagenous portion of the molecule is in the triple helical configuration prior to proteolytic excision from the parent molecule. The amino acid composition is remarkable when compared to tropocollagen in the large quantity of acidic residues, decreased glycine and imino acids, and the presence of cystcine. Three models of procollagen I structure are presented and discussed relative to the available experimental evidence.     

Polymeric collagen fibrils. An example of substrate-mediated steric obstruction of enzymic digestion.

Polymeric collagen fibrils have been reacted with fluorescein and rhodamine isothiocyanates to produce fluorescent dye-labelled fibrils, containing seven dye substituents per molecule of tropocollagen within the polymeric collagen fibrils. Two dye-labelled peptides per molecule of tropocollagen were solubilised by trypsin (EC 3.4.21.4) from the telopeptide regions and four dye-labelled peptides were located in the helical regions solubilised by bacterial collagenase (EC 3.4.24.3). The solubilisation of dye-labelled peptides from these insoluble substrates were employed to measure the kinetics of trypsin and collagenase digestion of the telopeptide and helical regions, respectively, of the insoluble polymeric collagen fibrils. These studies demonstrated an apparent excess of enzyme for the readily available substrate under conditions when it was known that a vast excess of substrate existed in the reaction mixture calculated in terms of a molecular ratio. A point of equivalence was established for both trypsin and bacterial collagenase, approximately one enzyme molecule per 870 substrate molecules. On either side of this point the quantity of products formed was controlled by either the enzyme concentration or the substrate concentration. The results can be explained in terms of the inaccessibility of tropocollagen molecules within the molecular architecture of the polymeric collagen fibrils. The external layer of tropocollagen molecules obstruct collagenolytic enzymes penetrating to, and forming enzyme-substrate complexes with, the bulk of the substrate within the interior of the fibrils.     

Biochemical and physiochemical characterization of pepsin-solubilized type-II collagen from bovine articular cartilage.

Solubilization of collagen from bovine articular with pepsin requires the preliminary extraction of proteoglycans from the ground substance. Biochemical and physiochemical properties of this pepsin-solubilized collagen are independent of the pretreatment (extraction with 1.5M-CaCl2, 5M-guanidinium chloride or 0.2M-NaOH) and of the age range (2-4-year-old and 2-month-old animals). Characterization of the de-natured components, of the CNBr peptides and of the amino acid and cross-link composition shows that the collagen of the hyaline cartilage is all type II. Electrical birefringence measurements showed the presence of tropocollagen molecules (length 280nm) and molecules whose length is slightly less than twice that of the tropocollagen molecules. This latter molecule may be a dimer composed of two monomers linked by intermolecular head-to-tail bonds and whose theoretical length (530nm), according to the quarter-stagger theory, is in good agreement with our measured values (510-530nm). We have verified that the beta-components of this collagen are formed of two alpha-chains linked by the stable intermolecular bond, dehydrodihydroxylysinonorleucine. These dimeric molecules are absent from solutions of skin collagen whose beta-components possess only aldol-type intramolecular cross-links. Although reconstituted fibres from solutions of skin and cartilage collagen are similar, the segment-long spacing crystallites formed with pepsin-solubilized cartilage collagen present a symmetrical and dimeric form corresponding to the lateral aggregation of two monomers with an overlap (90nm) of the C-terminal ends.     

Ultrastructural organization of heterochromatin within sea urchin sperm nuclei

Chromatin within swollen or lysed isolated sperm nuclei of the sea urchin, Strongylocentrotus purpuratus, was examined by electron microscopy. Spread preparations of lysed sperm nuclei demonstrated dense aggregates of nondispersed material and beaded filaments radiating from these aggregates. These beaded fibers are similar in size and appearance to the “beads-on-a-string” seen as characteristic of chromatin spreads from numerous interphase nuclei. The beads are nucleosomes that have an average diameter of 130 Å. The interconnecting string is 40 Å indiameter and corresponds to the spacer DNA. In thin sections of swollen nuclei the sperm chromatin appears to be composed of 400 Å superbeads that are closely apposed to form 400 Å fibers. As the chromatin disperses, the superbeads are seen to be attached to one another by chromatin fibers of 110 Å diameter. In thin sections, the 400 Å superbeads appear to disperse directly into the 110 Å fibers with no intervening structures. This work demonstrates that the heterochromatin in Strongylocentrotus purpuratus sperm nuclei is composed of nucleosomes that form 100 Å filaments that are compacted into 400 Å superbeads. The superbeads coalesce to give the morphological appearance of 400 Å fibers.     

Characterization of the amino-terminal segment in type III procollagen.

Native type III collagen and procollagen were prepared from fetal bovine skin. Examination of the cleavage products produced by digestion with tadpole collagenase demonstrated that the three palpha1(III) chains of type III procollagen were linked together by disulfide bonds occurring at both the amino-terminal and carboxy-terminal portions of the molecule. Type III collagen contained interchain disulfide bonds only in the carboxy-terminal region of the molecule. After digestion of procollagen with bacterial collagenase an amino-terminal, triple-stranded peptide fragment was isolated. The reduced and alkylated chain constituents of this fragment had molecular weights of about 21 000. After digestion of procollagen with cyanogen bromide a related triple-stranded fragment was isolated. The chains of the cyanogen bromide fragment had a molecular weight of about 27 000. When the collagenase-derived peptide was fully reduced and alkylated, it became susceptible to further digestion with bacterial collagenase. This treatment released a fragment of about 97 amino acid residues which contained 12 cystein residues and had an amino acid composition typical for globular proteins. A second, non-helical fragment of about 48 amino acid residues contained three cysteines. This latter fragment is formed from sequences that overlap the amino-terminal region in the collagen alpha1(III) chain by 20 amino acids and possesses an antigenic determinant specific for the alpha1(III) chain. The collagenase-sensitive region exposed by reduction comprised about 33 amino acid residues. It was recovered as a mixture of small peptides. These results indicate that the amino-terminal region of type III procollagen has the same type of structure as the homologous region of type I procollagen. It consists of a globular, a collagen-like and a non-helical domain. Interchain disulfide bonding and the occurrence of cysteines in the non-helical domain are, however, unique for type III procollagen.     

Shape and assembly of type IV procollagen obtained from cell culture.

Type IV procollagen was isolated from the culture medium of the teratocarcinoma cell line PYS-2 by affinity chromatography on heparin-Sepharose. Immunological studies showed that type IV procollagen is composed of pro-alpha 1(IV) and pro-alpha 2(IV) chains and contains two potential cross-linking sites which are located in the short triple-helical 7S domain and the globular domain NC1 . The 7S domain was also identified as the heparin binding site. Rotary shadowing visualized type IV procollagen as a single triple-helical rod (length 388 nm) with a globule at one end. Some of the procollagen in the medium, however, had formed aggregates by alignment of 2-4 molecules along their 7S domains. After deposition in the cell matrix, non-reducible cross-links between the 7S domains are formed while the globules of two procollagen molecules connect to each other. The latter may require a slight proteolytic processing of the globular domains NC1 . The shape of type IV procollagen and the initial steps in its assembly are compatible with a recently proposed network of type IV collagen molecules in basement membranes. Since both type IV collagen and laminin bind to heparin, the formation of higher ordered structures by interaction of both proteins with heparan-sulfate proteoglycan may occur in situ.     

Morphological and structural studies of poly(gly-gly-ala)

The ultrastructural morphology and x-ray and electron diffraction of poly (Gly-Gly-Ala) have been studied. The polymer has two forms; the first, form I possesses a super-folded cross-β structure, long fibers of which show some twisting and intertwining. Form II precipitates in a less distinct fibrous form from aqueous solution. The x-ray diffraction and oriented electron diffraction data suggest that form II is a polyglycine II helix situated in a monoclinic cell with dimensions a = 8.86 Å, b = 22.0 Å, c = 9.42 Å, and β = 90°. Combined with the morphological evidence it appears likely that form II is also in an antiparallel superfolded array.     

Fibrous long spacing collagen ultrastructure elucidated by atomic force microscopy.

Fibrous long spacing collagen (FLS) fibrils are collagen fibrils in which the periodicity is clearly greater than the 67-nm periodicity of native collagen. FLS fibrils were formed in vitro by the addition of alpha1-acid glycoprotein to an acidified solution of monomeric collagen and were imaged with atomic force microscopy. The fibrils formed were typically approximately 150 nm in diameter and had a distinct banding pattern with a 250-nm periodicity. At higher resolution, the mature FLS fibrils showed ultrastructure, both on the bands and in the interband region, which appears as protofibrils aligned along the main fibril axis. The alignment of protofibrils produced grooves along the main fibril, which were 2 nm deep and 20 nm in width. Examination of the tips of FLS fibrils suggests that they grow via the merging of protofibrils to the tip, followed by the entanglement and, ultimately, the tight packing of protofibrils. A comparison is made with native collagen in terms of structure and mechanism of assembly.     

Proteolysis of procollagen I.

1. Digestion of procollagen I which trypsin, pepsin or pronase performed at 20 degrees C causes the release of acidic non-collagenous fragments and hydroxyproline-rich fraction. Enzymatic proteolysis performed at 41 degrees C (above the temperature of denaturation) results in degradation of procollagen I to low-molecular peptides. 2. The hydroxyproline-rich fraction obtained by limited proteolysis of procollagen I with pepsin (at 20 degrees C) contains a material corresponding to alpha and beta subunits of tropocollagen. Reduction of the hydroxyproline-rich fraction released by trypsin or pronase (at 20 degrees C) causes the appearance of polypeptides similar to pro-alpha subunits.     

The structure of a COPII tubule

Nearly a third of all eukaryotic proteins are transported from the ER to the Golgi apparatus through the secretory pathway using COPII coated vesicles. Evidence suggests that this transport occurs via 500–900 Å vesicles that bud from the ER membrane. It has been shown that procollagen molecules utilize the COPII proteins for transport, but it is unclear how the COPII coat can accommodate these ～3000 Å long molecules. We now present a cryogenic electron tomographic reconstruction of a Sec13/31 tubule that is approximately 3300 Å long containing a hollow cylindrical interior that is 300 Å in diameter, dimensions that are consistent with those that are required to encapsulate a procollagen molecule wrapped in a membrane and accessory COPII components. This structure suggests a novel mechanism that the COPII coat may employ to transport elongated cargo.     

Secondary structure and interfacial aggregation of amyloid-beta(1-40) on sodium dodecyl sulfate micelles

Alzheimer's disease (AD) is characterized by the presence of large numbers of fibrillar amyloid deposits in the form of senile plaques in the brain. The fibrils in senile plaques are composed of 40- and 42-residue amyloid-beta (Abeta) peptides. Several lines of evidence indicate that fibrillar Abeta and especially soluble Abeta aggregates are important in the pathogenesis of AD, and many laboratories have investigated soluble Abeta aggregates generated from monomeric Abeta in vitro. Of these in vitro aggregates, the best characterized are called protofibrils. They are composed of globules and short rods, show primarily beta-structure by circular dichroism (CD), enhance the fluorescence of bound thioflavin T, and readily seed the growth of long fibrils. However, one difficulty in correlating soluble Abeta aggregates formed in vitro with those in vivo is the high probability that cellular interfaces affect the aggregation rates and even the aggregate structures. Reports that focus on the features of interfaces that are important in Abeta aggregation have found that amphiphilic interactions and micellar-like Abeta structures may play a role. We previously described the formation of Abeta(1-40) aggregates at polar-nonpolar interfaces, including those generated at microdroplets formed in dilute hexafluoro-2-propanol (HFIP). Here we compared the Abeta(1-40) aggregates produced on sodium dodecyl sulfate (SDS) micelles, which may be a better model of biological membranes with phospholipids that have anionic headgroups. At both HFIP and SDS interfaces, changes in peptide secondary structure were observed by CD immediately when Abeta(1-40) was introduced. With HFIP, the change involved an increase in predominant beta-structure content and in fluorescence with thioflavin T, while with SDS, a partial alpha-helical conformation was adopted that gave no fluorescence. However, in both systems, initial amorphous clustered aggregates progressed to soluble fibers rich in beta-structure over a roughly 2 day period. Fiber formation was much faster than in the absence of an interface, presumably because of the close intermolecular proximity of peptides at the interfaces. While these fibers resembled protofibrils, they failed to seed the aggregation of Abeta(1-40) monomers effectively.     

X‐ray structure of Danio rerio secretagogin: A hexa‐EF‐hand calcium sensor

Many essential physiological processes are regulated by the modulation of calcium concentration in the cell. The EF‐hand proteins represent a superfamily of calcium‐binding proteins involved in calcium signaling and homeostasis. Secretagogin is a hexa‐EF‐hand protein that is highly expressed in pancreatic islet of Langerhans and neuroendocrine cells and may play a role in the trafficking of secretory granules. We present the X‐ray structure of Danio rerio secretagogin, which is 73% identical to human secretagogin, in calcium‐free form at 2.1‐Å resolution. Secretagogin consists of the three globular domains each of which contains a pair of EF‐hand motifs. The domains are arranged into a V‐shaped molecule with a distinct groove formed at the interface of the domains. Comparison of the secretagogin structure with the solution structure of calcium‐loaded calbindin D_28K revealed a striking difference in the spatial arrangement of their domains, which involves ～180° rotation of the first globular domain with respect to the module formed by the remaining domains. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Mechanisms of the self-assembly of EAK16-family peptides into fibrillar and globular structures: molecular dynamics simulations from nano- to micro-seconds

The self-assembly of EAK16-family peptides in a bulk solution was studied using a combination of all-atom and coarse-grained molecular dynamics simulations. In addition, specified concentrations of EAK16 peptides were induced to form fibrillary or globular assemblies in vitro. The results show that the combination of all-atom molecular dynamics simulations on the single- and double-chain levels and coarse-grained simulations on the many-chain level predicts the experimental observations reasonably well. At neutral pH conditions, EAK16-I and EAK16-II assemble into fibrillary structures, whereas EAK16-IV aggregates into globular assemblies. Mechanisms of the formation of fibrillar and globular assemblies are described using the simulation results.     

Observations on the different substrate behavior of tropocollagen molecules in solution and intermolecularly cross-linked tropocollagen within insoluble polymeric collagen fibrils.

Bacterial collagenase was used to compare the extent of digestion of tropocollagen monomers in solution and in reconstituted fibrils with that of tropocollagen molecules intermolecularly cross-linked within insoluble polymeric collagen fibrils obtained from mature tendons at given time-intervals. The extent of digestion of tropocollagen monomers in solution was directly proportional to the enzyme concentration (a range of enzyme substrate molar ratios 1:200 to 1:10 was used). The extent of digestion of polymeric collagen was followed by measuring the solubilization of fluorescent peptides from fluorescent-labelled insoluble polymeric collagen fibrils. The extent of digestion of tropocollagen within polymeric collagen was linear over a very small range of enzyme concentrations, when the enzyme/substrate ratio in the reaction mixture was less than 1:400 on a molecular basis. The behavior of tropocollagen in the form of reconstituted collagen fibrils, which had been matured at 37 degrees C for 8 weeks, was intermediate between the behaviour of solutions of tropocollagen and insoluble polymeric collagen fibrils. The significance of the results is discussed in terms of the structure of polymeric collagen fibrils and the protection against enzymic attack provided by tropocollagen molecules on the circumference of the fibril. The results suggest that assays of collagenase activities based on tropocollagen as substrate cannot be directly related to the ability of these enzymes to degrade mature insoluble collagen fibrils.