Basement membranes: molecular organization and function in development and disease.
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Animals↗
Biomedical subjects
P D Yurchenco
Publications and source records attributed to P D Yurchenco.
Type IV collagen lateral associations in the EHS tumor matrix. Comparison with amniotic and in vitro networks.
The macromolecular structural organization of the type IV collagen network in the extracellular matrix of the EHS tumor has been investigated using a stereoscopic freeze-dry Pt/C replication technique. This network, which can be specifically decorated with type IV collagen antibody, is formed in great part by the lateral joining of narrow filaments (2.7 nm average metal coated diameter) to form a complex three-dimensional irregular polygonal array of variable diameter branching strands. Globular domains, similar to the C-terminal globular domains of purified type IV dimers, can be identified in the network. In many regions of the network the filaments appear to twist around each other along the strand axis. The network is similar to that visualized in the human amnion as well as to a reconstituted network formed in vitro. These data strongly suggest that the laterally and end-domain-associated network is a widespread supramolecular architecture of type IV collagen in basement membranes.
Amnion↗
Self-assembly of a high molecular weight basement membrane heparan sulfate proteoglycan into dimers and oligomers.
A high molecular weight basement membrane heparan sulfate proteoglycan, isolated from murine Englebreth-Holm-Swarm tumor, is seen in platinum replicas as an elongated flexible core (Mr = 450,000) consisting of a series of tandem globular domains from which extend, at one end, two to three heparan sulfate chains (average Mr = 80,000 each). This macromolecule will self-assemble into dimers and lesser amounts of oligomers when incubated in neutral isotonic buffer. These molecular species can be separated by zonal velocity sedimentation and assembly is seen to be time- and concentration-dependent. In rotary-shadowed platinum replicas the binding region is found at or near the end of the core at the pole opposite the origin of the heparan sulfate chains. Dimers are double-length structures and oligomers are seen as stellate clusters: in both, the heparan sulfate chains appear peripherally oriented. While isolated cores self-assemble, isolated heparan sulfate chains do not bind intact proteoglycans. Furthermore, proteolytic removal of a non-heparan sulfate containing core moiety destroys the ability of the proteoglycan monomer to form larger species or bind intact proteoglycan, further supporting the binding topography determined morphologically. These negatively charged macromolecular complexes may be important contributors to basement membrane structure and function.
Animals↗
Structure of low density heparan sulfate proteoglycan isolated from a mouse tumor basement membrane.
A large heparan sulfate proteoglycan of low buoyant density (p = 1.32 to 1.40 g/cm3 in 6 M-guanidine.HCl) was extracted from a tumor basement membrane with denaturing solvents and purified by chromatography and CsCl gradient centrifugation. Chemical, immunological, physical and electron microscopical analyses have demonstrated a high degree of purity and have allowed us to propose a structural model for this proteoglycan. It is composed of an 80 nm long protein core formed from a single polypeptide chain (Mr about 500,000) with intrachain disulfide bonds. This core is folded into a row of six globular domains of variable size as shown by electron microscopy after rotary shadowing and negative staining. A multidomain structure was confirmed by protease digestion experiments that allowed the isolation of a single heparan sulfate-containing peptide segment representing less than 5% of the total mass of the protein core. Electron microscopy has visualized generally three heparan sulfate chains in each molecule close to each other at one pole of the protein core. The molecular mass and length (100 to 170 nm) of the heparan sulfate chains were found to vary consistently between different preparations. The mass per length ratio (350 nm-1) indicated an extended conformation for the heparan sulfate side-chains. These structural features are distinctly different from those of the high density proteoglycan, suggesting that both forms of basement membrane heparan sulfate proteoglycan are genetically distinct and not derived from a common precursor.
Amino Acids↗
Basement membrane structure in situ: evidence for lateral associations in the type IV collagen network.
To determine molecular architecture of the type IV collagen network in situ, the human amniotic basement membrane has been studied en face in stereo relief by high resolution unidirectional metal shadow casting aided by antibody decoration and morphometry. The appearance of the intact basement membrane is that of a thin sheet in which there are regions of branching strands. Salt extraction further exposes these strands to reveal an extensive irregular polygonal network that can be specifically decorated with gold-conjugated anti-type IV collagen antibody. At high magnification one sees that the network, which contains integral (9-11 nm net diameter) globular domains, is formed in great part by lateral association of monomolecular filaments to form branching strands of variable but narrow diameters. Branch points are variably spaced apart by an average of 45 nm with 4.4 globular domains per micron of strand length. Monomolecular filaments (1.7-nm net diameter) often appear to twist around each other along the strand axis; we propose that super helix formation is an inherent characteristic of lateral assembly. A previous study (Yurchenco, P. D., and H. Furthmayr. 1984. Biochemistry. 23:1839) presented evidence that purified murine type IV collagen dimers polymerize to form polygonal arrays of laterally as well as end-domain-associated molecules. The architecture of this polymer is similar to the network seen in the amnion, with lateral binding a major contributor to each. Thus, to a first approximation, isolated type IV collagen can reconstitute in vitro the polymeric molecular architecture it assumes in vivo.
Amnion↗
Mechanisms of cytoskeletal regulation: functional and antigenic diversity in human erythrocyte and brain beta spectrin.
A study of human erythrocyte and brain spectrin with particular emphasis on the beta subunits revealed a structural homology but functional dissimilarity between these two molecules. Six monoclonal antibodies raised to human erythrocyte beta spectrin identify three of the four proteolytically defined domains of erythrocyte beta spectrin. Five of these monoclonal antibodies cross-react with human brain spectrin. None of a previously identified set of alpha erythrocyte spectrin monoclonal antibodies [Yurchenco et al: J Biol Chem 257:9102, 1982] reacted with brain spectrin. A domain map generated by limited tryptic digestion shows that brain spectrin is composed of proteolytically resistant domains analogous to erythrocyte spectrin, but the brain protein is more basic. The binding of brain spectrin to erythrocyte ankyrin, both in solution and on erythrocyte IOVs, yielded an association constant approximately 100 time weaker than for erythrocyte spectrin. The binding of azido-calmodulin under native conditions was specific for the erythrocyte beta subunit but was not calcium dependent. In contrast, azido-calmodulin bound only to the alpha subunit of brain spectrin in a calcium-dependent manner. The similarity of structure but modified functional characteristics of the brain and erythrocyte beta spectrins suggest that these proteins serve different cellular roles.
Amino Acids↗
Models for the self-assembly of basement membrane.
Basement membranes contain a number of intrinsic macromolecular components which are unique to these structures and which cooperatively assemble into specific heteropolymeric matrices. Type IV collagen triple helical monomers bind together at their amino-terminal, carboxy-terminal, and lateral domains to form a lattice-like array. Laminin, in a two-step process, binds to itself at its terminal globular domains to form polymers and also binds collagen at two distinct sites along the collagen chain. Heparan sulfate proteoglycan has been found to bind both collagen and laminin, suggesting a reversible crosslinking function. On the basis of the data derived from self-association studies, it is possible to begin considering models for the assembly and structure of these ubiquitous matrices.
Animals↗
Laminin polymerization in vitro. Evidence for a two-step assembly with domain specificity.
Laminin, a major structural glycoprotein of basement membranes, has been found to self-associate in vitro into large polymers. The formation of these complexes can be followed by the development of turbidity upon incubation in neutral phosphate buffer at 21-35 degrees C and is seen to be time-, concentration-, and temperature-dependent. The process is thermally reversible at 4 degrees C and the protein can be cycled between a dispersed and an aggregated state by alternating between 4 and 35 degrees C. Following incubation at 35 degrees C much of the monomeric laminin, which sediments at 11.4 S, is now seen to sediment at greater than 25 S. Both by turbidometric and sedimentation analysis, an apparent critical concentration for assembly of about 0.1 mg/ml (10(-7) M) is observed and is interpreted as evidence for a nucleation-propagation polymerization mechanism. The relative paucity of intermediates seen in a size-distribution analysis lends further support for this model. On platinum replicas obtained by rotary shadowing analysis, mostly free monomers are seen in the cold while after incubation at 35 degrees C, large multimeric aggregates with smaller amounts of oligomers are observed. The interaction between individual molecules appears to be specific because the dimers, trimers, and smaller oligomers are only associated at the terminal globular domains of the laminin molecules. In addition, removal of the globular domains of laminin with pepsin, which yields fragment P1, abolishes self-association. A divalent cation dependency for polymerization can be demonstrated and incubation in the presence of EDTA stops the polymerization at an oligomeric intermediate step. Hence overall laminin self-assembly can be divided into at least two steps: an initial temperature-dependent, divalent cation independent step followed by a divalent cation-dependent step.
Animals↗
Binding of laminin to type IV collagen: a morphological study.
A mixture of laminin and type IV collagen was analyzed by rotary shadowing using carbon/platinum and electron microscopy. Laminin was found to form distinct complexes with type IV collagen: one site of interaction is located 140 nm from the COOH-terminal, noncollagenous (NC1) domain and the other is located within the NH2-terminal region. The isolated NC1 fragment of type IV collagen does not appear to interact with laminin, while pepsin-treated type IV collagen, which lacks the NC1 domain, retains its ability to form complexes with laminin. Analysis of the laminin-type IV complexes indicates that laminin binds to type IV collagen via the globular regions of either of its four arms. This finding is supported by experiments using fragment P1 of laminin which lacks the globular regions and which does not bind to type IV collagen in a specific way. In addition, after heat-denaturation of laminin no specific binding is observed.
Animals↗
Self-assembly of basement membrane collagen.
The in vitro self-assembly of murine type IV collagen was examined by using biochemical and morphological techniques. Dimeric collagen undergoes a rapid and reversible thermal gelation at neutral pH without an appreciable lag period. The process is seen to be concentration dependent and inhibited by 2 M urea. The formed complex can be visualized by electron microscopy rotary shadowing as an irregular polygonal lattice network with extensive side by side associations within the collagenous triple-helical part of the molecules, two and three strands thick. Measurements on the matrix suggest a median stagger dimension of 170 nm, one-fifth the length of a dimer. The conversion of pepsin-generated monomers into N-terminally bound tetramers can also be demonstrated in vitro. This process is also concentration dependent and inhibited and reversed by 2 M urea but is thermally irreversible and occurs at a slow rate relative to the lateral associations. These tetramers can be seen by rotary shadowing as four-armed "spider" structures. It is proposed that lateral associations, by virtue of their faster rate of formation, precede 7S bond formation, and several models for the assembly of basement membrane collagen are discussed.
Animals↗
The ultrastructural organization and architecture of basement membranes.
Basement membranes are ubiquitous complex, multicomponent structures having diverse functions. They are morphologically distinct and exhibit specific structural details including the lamina rara and lamina densa. In addition, the interstitial stroma abutting the lamina densa has a unique organization. While the composition of basement membranes is still incompletely known, several components have been identified, including collagen types IV and V, laminin and heparan sulphate proteoglycan. High resolution immunoelectron microscopic studies have allowed the development of various models of the organization and architecture of the basement membrane, suggesting specific localizations of the various collagen types and specific domains of the collagen molecules, laminin and other components. In addition, high resolution metal shadow casting techniques have allowed the development of molecular models of specific components of the basement membrane and methods of studying the domain structure and interactions of these components.
Animals↗
Structure of human erythrocyte spectrin. I. Isolation of the alpha-I domain and its cyanogen bromide peptides.
The alpha-I domain of human erythrocyte spectrin was produced by a mild tryptic digestion of the intact molecule and purified by a single step affinity chromatography procedure using a monoclonal antibody. A tryptic peptide representing the alpha-I domain, which migrated on polyacrylamide gels as an 80,000-dalton peptide, was subjected to automated Edman-Begg degradation. Products from automated sequencing were identified by reverse-phase high performance liquid chromatography. Two smaller proteolytic products of the alpha-I domain (T74 and T50) were also subjected to automated sequence analysis. CNBr cleavage of the alpha-I domain produced nine unique peptides which were separated by gel filtration on a high performance liquid chromatograph. Peptides were further purified by reverse-phase chromatography and characterized by amino acid analysis. Partial sequences were determined by automated NH2-terminal sequence analysis. A single aspartate-proline bond, which was partially hydrolyzed during the cyanogen bromide cleavage reaction, was also identified. These sequence data include the first 86 residues of the alpha-I domain, and the spectrin oligomer binding site has been tentatively localized within the first 39 residues. The sequence of 293 residues of a total 633 residues in the alpha-I domain is presented and represents the first structural information for this protein.
Amino Acid Sequence↗
Monoclonal antibodies as probes of domain structure of the spectrin alpha subunit.
A library of nine monoclonal antibodies which bind to the alpha subunit of human erythrocyte spectrin has been established. The specificity of these antibodies confirms the alignment and uniqueness of each of the five previously identified peptide domains in this subunit and establishes the identity of additional smaller proteolytic peptide fragments. This immunochemical approach is complementary to the identification of peptide relationships by two-dimensional chymotryptic peptide mapping, and the results of both methods are in complete agreement.
Antibodies, Monoclonal↗
Expression of red cell membrane proteins in erythroid precursor cells.
Specific antibodies to human glycophorin A and spectrin were used to study the expression of these membrane proteins in normal and pathologic human bone marrow. In immunofluorescence experiments spectrin and glycophorin A are found in 50-60% of the nucleated cells in normal bone marrow. These two proteins are expressed at all stages of red cell differentiation and can be traced at least to the earliest morphologically recognizable nucleated red cell precursor, the proerythroblast; the two proteins are specific for cells of the red cell series and are not found to be expressed in lymphocytic, granulocytic cells or platelets. These conclusions were drawn from studies on bone marrow in patients with a temporary block in erythropoiesis at the level of stem cells or of the pronormoblast. Bone marrow from these individuals either lacked all nucleated cells stainable for glycophorin A and spectrin or contained only pronormoblasts. Similar findings were obtained on spleen cells from mice which were made severely anemic by multiple injections with N-acetyl-phenylhydrazine. Antibodies to a sialoglycoprotein isolated from mouse red cell membranes stain 70-80% of all cells in the spleen of anemic animals, while only 1-2% of such cells are seen in the spleen of normal animals. Spectrin and glycophorin A could be labeled metabolically and isolated using specific antibodies. The human tumor cell line K562 expresses both membrane proteins, but induction experiments with various agents thus far have failed to change their expression.
Adult↗
Labeling complex carbohydrates of animal cells with monosaccharides.
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Animals↗
Equilibration of fucosyl glycoprotein pools in HeLa cells.
The pool sizes, label equilibration times, and specific radioactivity relationships of fucosyl glycoproteins and precursors have been examined in exponentially growing HeLa S3 cells (generation time about 23 h) using a quantitative radioisotopic approach. The specific radioactivity of the precursor GDP-fucose (pool size 0.52 +/- 4% nmol/10(7) cells) equilibrates with radioactive fucose in the medium in about 1 h. 10(7) cells contain 5.3 +/- 16% nmol of glycoprotein fucose of which 96-98% resides in or on the cell surfaces and is equilibrated isotopically within 22 h of labeling; 2% or less is in an internal pool, some of which is precursor to plasma membranes and some of which is released as soluble glycoprotein directly to the medium without random mixing with the plasma membrane glycoprotein. Because we cannot rule out the presence of internal free fucose, 2% of the total glycoprotein fucose could be in the degradative pathway being recycled internally before release of free fucose. In rate terms and in a particular culture where 10(7) cells contained 4.4 nmol of glycoprotein fucose, a total of 11.1 nmol of glycoprotein fucose is synthesized per generation (9-11% of the total cell glycoprotein fucose/h). Of this, 2.5 nmol of glycoprotein fucose per generation is released directly into the growth medium without mixing with the plasma membrane glycoprotein fucose. The small internal pool feeding glycoprotein fucose to the plasma membrane does so at the rate of 8.6 nmol/10(7) cells per generation, 4.2 nmol per generation of which, after mixing with the plasma membrane glycoprotein fucose, is ultimately released into the growth medium, 75-80% as free frucose. This release process is independent of cell density and the presence of serum in the growth medium.
Cell Fractionation↗
Fucosyl-glycoprotein and precursor polls in HeLa cells.
An enzymatic-radioactive isotope method has been developed for the direct quantitation of L-fucose in amounts as low at 0.5 plus or minus 0.05 nmol. Fucose kinase is used to transfer [32-P]phosphate from ATP to [3-H]fucose. The labeled enzymatic products are then separated electrophoretically and the amount and specific activity of the fucose are determined from the known specific activity of the phosphate donor. This assay has been used to measure the GDP-L-fucose and macromolecualar fucose in HeLa cells after extraction and purification of the sugar. It has been determined there are 0.5 nmol of GDP-L-fucose in 10-7 cells with a nine- to tenfold dilution of specific activity in converting L-[3-H] fucose to GDP-L-[3-H]fucose. After 2 to 3 days of labeling, the GDP-L-[3-H]fucose pool is essentially at equilibrium with the macromolecular pool, and hence it can be concluded that the dilution of label is due to a nine- to tenfold contribution to GDP-L-fucose from an endogenous source, as compared to exogenously supplied fucose. The fucosyl-glycoprotein pool has been shown to be much larger containing 6 to 8 nmol of fucose in 10-7 cells. It has further been shown that GDP-fucose is the only soluble fucose intermediate present in significant amount.
Cell Division↗