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Lars-Åke Fransson - One of the best experts on this subject based on the ideXlab platform.
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Initiation of Galactosaminoglycan biosynthesis
European journal of biochemistry, 1999Co-Authors: Jonatan Moses, Åke Oldberg, Lars-Åke FranssonAbstract:By using various radiolabelled precursors, glycosylation and phosphorylation of decorin in a rat fibroblast cell line was investigated in the presence of increasing concentrations of p-nitrophenyl-O-β-d-xylopyranoside. Decorin core protein glycanation was suppressed to ≈25% of the normal level in the presence of 2 mm and 3 mm xyloside. Glycans/saccharides were released from the core protein and size-separated by gel chromatography. The intracellular decorin obtained from cells treated with 2 mm xyloside was substituted with Xyl and also with Gal-Xyl and Gal-Gal-Xyl, but not with longer saccharides. Only the trisaccharide contained an almost fully phosphorylated Xyl. We conclude that galactosylation of endogenous, xylosylated decorin and exogenous xyloside probably follow separate pathways or that xylosides and early decorin glycoforms are kept separated. At the addition of the first glucuronic acid the two pathways seem to merge and dephosphorylation of decorin takes place. Xyloside-primed and secreted Galactosaminoglycan chains produced simultanously retained phosphorylated Xyl. Inadequate dephosphorylation could be due to excess substrate or to a short transit.time. As shown previously [Moses, J., Oldberg, A., Eklund, E. & Fransson, L.-A. (1997) Eur. J. Biochem.248, 767–774], brefeldin A-arrested decorin is substituted with the linkage-region extended with an undersulphated and incomplete Galactosaminoglycan chain. In cells treated with this drug, xylosides were unable to prime Galactosaminoglycan synthesis and unable to inhibit glycosylation and phosporylation of decorin.
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Biosynthesis of the proteoglycan decorin Identification of intermediates in Galactosaminoglycan assembly
European journal of biochemistry, 1997Co-Authors: Jonatan Moses, Åke Oldberg, Erik A. Eklund, Lars-Åke FranssonAbstract:Biosynthesis of decorin was investigated by incubating a rat fibroblast cell line with various radio-labelled protein and Galactosaminoglycan precursors. The following cell-associated and distinct intermediates were isolated and identified: a pool of non-glycosylated core protein, two pools of decorin with incomplete chains, one with three sulphated disaccharide repeats and another with five or more sulphated disaccharide repeats, as well as decorin with mature chains. Results of pulse/chase experiments indicated that these pools represented discrete stages in chain growth. Treatment with brefeldin A, which blocks transport from the endoplasmic reticulum to the Golgi, resulted in accumulation of decorin with an incomplete chain containing six or seven largely unsulphated disaccharide repeats. During recovery from drug treatment, 4-sulfation reappeared earlier than 6-sulfation. The results suggest that the Galactosaminoglycan assembly-line consists of separate multienzyme complexes that build only a limited section of the chain. Furthermore, brefeldin A causes segregation of compartments involved in separate stages of the assembly line. In an earlier report [Moses, J., Oldberg, A., Cheng, F. & Fransson, L.-A. (1997) Eur. J. Biochem. 248, 521–526] we took advantage of such segregation to identify and characterize a transient 2-phosphorylation of xylose in the linkage region.
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Biosynthesis of the proteoglycan decorin Transient 2-phosphorylation of xylose during formation of the trisaccharide linkage region
European journal of biochemistry, 1997Co-Authors: Jonatan Moses, Åke Oldberg, Fang Cheng, Lars-Åke FranssonAbstract:Phosphorylation of decorin was investigated by incubating a rat fibroblast cell line with radiolabelled phosphate and carbohydrate precursors. There was a transient phosphorylation of the linkage-region saccharides in intracellular decorin prior to assembly of the Galactosaminoglycan chain. Phosphorylation gradually increased from xylosylated, galactosyl-xylosylated to galactosyl-galactosyl-xylosylated core protein where all trisaccharide stubs were phosphorylated. Addition of the first glucuronate residue was accompanied by rapid dephosphorylation. Brefeldin A treatment resulted in segregation of Galactosaminoglycan synthesis and dephosphorylation. Enzymatic degradation of brefeldin-A-arrested immature proteoglycan with incomplete Galactosaminoglycan chain [Moses, J., Oldberg, A., Eklund, E. & Fransson, L.-A. (1997) Eur. J. Biochem., in the press] by using chondroitin AC lyase and chondro-glycuronidase, followed by beta-galactosidase treatment, demonstrated the sequence galactosyl-galactosyl-phosphoxylose. The xylose was resistant to direct periodate oxidation, but sensitive after treatment with alkaline phosphatase, showing that the phosphate was located at C2 of xylose. The transient 2-phosphorylation of xylose may be involved in intracellular transport and/or in the control of modifications of the glycan chain.
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Amino‐terminal deletions in the decorin core protein leads to the biosynthesis of proteoglycans with shorter glycosaminoglycan chains
FEBS letters, 1996Co-Authors: Åke Oldberg, Jonatan Moses, Per Antonsson, Lars-Åke FranssonAbstract:Analysis of the N-terminal sequence of decorin purified from connective tissues and comparison with the sequence deduced from the cDNA indicate that the nascent proteoglycan has a 14 amino acid residue N-terminal propeptide. Mammalian expression vectors encoding wild-type decorin and decorin with deletions in the propeptide were used to transform COS and CHO cells. Cells transformed with vectors encoding deletion variants of decorin synthesize proteoglycans with shorter Galactosaminoglycan chains than cells transformed with wild-type decorin. This effect on the polysaccharide chain length may be due to a lower affinity between the core protein and the glycosyltransferases synthesizing the linkage region. Alternatively, the deletions may affect the intracellular transport of decorin. An antiserum prepared against the N-terminal propeptide immunoprecipitated decorin secreted by cultured cells, showing that decorin is exported with the N-terminal region intact.
Meenakshi Sthanam - One of the best experts on this subject based on the ideXlab platform.
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perlecan and basement membrane chondroitin sulfate proteoglycan bamacan are two basement membrane chondroitin dermatan sulfate proteoglycans in the engelbreth holm swarm tumor matrix
Journal of Biological Chemistry, 1996Co-Authors: John R. Couchman, Rajesh Kapoor, Meenakshi SthanamAbstract:Abstract The presence of proteoglycans bearing Galactosaminoglycan chains has been reported, but none has been identified previously in the matrix of the Engelbreth-Holm-Swarm tumor, which is a source of several basement membrane components. This tumor matrix contains perlecan, a large, low buoyant density heparan sulfate proteoglycan, widespread in many basement membranes and connective tissues. We now identify two distinct proteoglycan species from this tumor source, which are substituted with Galactosaminoglycans and which show basement membrane localization by immunohistochemistry. One species is perlecan but, in addition to being present as a heparan sulfate proteoglycan, it is also present as a hybrid molecule, with dermatan sulfate chains. A minor population of perlecan apparently lacks heparan sulfate chains totally, and some of this is substituted with chondroitin sulfate. The second species is immunologically related to basement membrane-chondroitin sulfate proteoglycan (BM-CSPG) and bears chondroitin sulfate chains. No BM-CSPG was detectable which was substituted with heparan sulfate chains. A combination of immunological and molecular approaches, including cDNA cloning, showed that perlecan and BM-CSPG are distinct in core protein structure. Both are, however, basement membrane components, although there are tissue-specific differences in their distribution.
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Perlecan and Basement Membrane-Chondroitin Sulfate Proteoglycan (Bamacan) Are Two Basement Membrane Chondroitin/Dermatan Sulfate Proteoglycans in the Engelbreth-Holm-Swarm Tumor Matrix
The Journal of biological chemistry, 1996Co-Authors: John R. Couchman, Rajesh Kapoor, Meenakshi SthanamAbstract:Abstract The presence of proteoglycans bearing Galactosaminoglycan chains has been reported, but none has been identified previously in the matrix of the Engelbreth-Holm-Swarm tumor, which is a source of several basement membrane components. This tumor matrix contains perlecan, a large, low buoyant density heparan sulfate proteoglycan, widespread in many basement membranes and connective tissues. We now identify two distinct proteoglycan species from this tumor source, which are substituted with Galactosaminoglycans and which show basement membrane localization by immunohistochemistry. One species is perlecan but, in addition to being present as a heparan sulfate proteoglycan, it is also present as a hybrid molecule, with dermatan sulfate chains. A minor population of perlecan apparently lacks heparan sulfate chains totally, and some of this is substituted with chondroitin sulfate. The second species is immunologically related to basement membrane-chondroitin sulfate proteoglycan (BM-CSPG) and bears chondroitin sulfate chains. No BM-CSPG was detectable which was substituted with heparan sulfate chains. A combination of immunological and molecular approaches, including cDNA cloning, showed that perlecan and BM-CSPG are distinct in core protein structure. Both are, however, basement membrane components, although there are tissue-specific differences in their distribution.
Hans Kresse - One of the best experts on this subject based on the ideXlab platform.
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core protein dependence of epimerization of glucuronosyl residues in Galactosaminoglycans
Journal of Biological Chemistry, 2002Co-Authors: Daniela G. Seidler, Egon Breuer, Jane K Grandeallen, Vincent C Hascall, Hans KresseAbstract:Chondroitin sulfate and dermatan sulfate proteoglycans are distinguished by differences in their proportion of d-glucuronosyl and l-iduronosyl residues, the latter being formed by chondroitin-glucuronate 5-epimerase during or after glycosaminoglycan chain polymerization. To investigate the influence of the core protein on the extent of epimerization, we expressed chimeric proteins in 293 HEK cells constructed from intact or modified Met(1)-Gln(153) of decorin (DCN), which normally has a single dermatan sulfate chain at Ser(34), in combination with intact or modified Leu(241)-Ser(353) of CSF-1, which has a chondroitin sulfate attachment site at Ser(309). Transfected DCN(M1-Q153), like full-length DCN, contained approximately 20% l-iduronate. Conversely, transfected CSF-1(L241-S353), attached C-terminally on the DCN prepropeptide, contained almost exclusively d-glucuronate. Transfected intact chimeric DCN(M1-Q153)-CSF-1(L241-S353), with two glycosaminoglycan chains, also contained almost exclusively d-glucuronate in chains at both sites, as did chimeras in which alanine was substituted for serine at either of the glycosaminoglycan attachment sites. Nevertheless, undersulfated intact chimeric proteoglycan was an effective substrate for epimerization of glucuronate to iduronate residues when incubated with microsomal proteins and 3'-phosphoadenylylphosphosulfate. C-terminal truncation constructs were prepared from the full-length chimera with an alanine substitution at the CSF-1 glycosaminoglycan attachment site. Transfected truncations retaining the alanine-blocked site contained chains with essentially only glucuronate, whereas those further truncated by 49 or more amino acids and missing the modified attachment site contained chains with approximately 15% iduronate. This 49-amino acid region contains a 7-amino acid motif that appears to be conserved in several chondroitin sulfate proteoglycans. The results are consistent with a model in which the core protein, possibly via this motif, is responsible for routing to subcellular compartments with or without sufficient access to chondroitin-glucuronate 5-epimerase for the addition of chains with or without iduronate residues, respectively.
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Chondroitin/Dermatan Sulphate Promotes the Survival of Neurons from Rat Embryonic Neocortex
The European journal of neuroscience, 1997Co-Authors: Joachim Kappler, Hans Kresse, Ulrich Junghans, Antje Koops, Christine C. Stichel, Heinz-j. Hausser, Hans Werner MüllerAbstract:Recently we have shown that biglycan, a small chondroitin sulphate proteoglycan of the extracellular matrix, supports the survival of cultured neurons from the developing neocortex of embryonic day 15 rats. Here we investigate the structure-function relationship of this neurotrophic proteoglycan and show that chondroitin/dermatan sulphate chains are the active moieties supporting survival. Heparin, a highly sulphated glucosaminoglycan, is less active than the Galactosaminoglycans (chondroitin-4-sulphate, chondroitin-6-sulphate and dermatan sulphate), whereas hyaluronic acid, an unsulphated glucosaminoglycan, does not support neuron survival. Galactosaminoglycans must be in direct contact with neurons to cause survival. Experiments with elevated potassium concentrations and antagonists of voltage-gated calcium channels exclude the involvement of membrane depolarization. However, genistein and an erbstatin analogue, which are inhibitors of tyrosine kinases with low specificity, abolished neuron survival in the presence of chondroitin/dermatan sulphate, whereas a selective inhibitor of neurotrophin receptor kinases (K252a) had no suppressive effect. Thus, yet unidentified tyrosine kinases are involved in the chondroitin/dermatan sulphate-dependent survival of neocortical neurons. In the embryonic stages of rat neocortical development chondroitin sulphate is mainly located in layers I, V and VI and the subplate. Chondroitin sulphate expression is maintained after birth, extends up to cortical layer IV on postnatal day 7, and is down-regulated until postnatal day 21 concomitant with the period of naturally occurring cell death. The latter observation is consistent with a putative role of chondroitin sulphate in the control of neuron survival during cortical histogenesis.
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Different Galactosaminoglycan composition of small proteoglycans from osteosarcoma cells.
Glycobiology, 1993Co-Authors: Heinz Hausser, Elke Schönherr, Hans KresseAbstract:The expression of the core proteins and the co-polymeric structure of the glycosaminoglycan chains of three different small proteoglycans (biglycan, decorin, proteoglycan-100) have been examined in the human osteosarcoma cell line MG-63. The three proteoglycans, which are carrying either one or two chondroitin/dermatan sulphate chains, were synthesized in a similar molar ratio, as determined by [35S]methionine as well as by [35S]sulphate incorporation. After sulphate ester formation, they were secreted into the culture medium with similar kinetics. Immune staining with monospecific antibodies revealed that at least biglycan and proteoglycan-100 were present in all individual cells. However, in contrast to these similarities, the glycosaminoglycan moiety of proteoglycan-100 was composed exclusively of chondroitin 4- and 6-sulphate repeating units, whereas biglycan and decorin contained hybrid polymers of chondroitin and dermatan sulphate with approximately 90% 4-sulphated disaccharide repeating units. Treatment with transforming growth factor-beta resulted in a marked down-regulation of proteoglycan-100 synthesis without significant alteration of its glycosaminoglycan structure. Up-regulation of biglycan and moderate down-regulation of decorin were accompanied by a small decrease in the conversion of chondroitin to dermatan sulphate disaccharide units in both cases. The specific stimulation of the biosynthesis of proteoglycan-100 by tumour necrosis factor-alpha was without consequence for its glycosaminoglycan composition. Treatment with tumour necrosis factor-alpha had no influence on the synthesis and glycosaminoglycan structure of biglycan and decorin. These findings support the proposal of the importance of the core protein for the determination of the extent of glycosaminoglycan modification.
Daniela G. Seidler - One of the best experts on this subject based on the ideXlab platform.
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The Galactosaminoglycan-containing decorin and its impact on diseases.
Current opinion in structural biology, 2012Co-Authors: Daniela G. SeidlerAbstract:Decorin, a member of the small leucine-rich proteoglycans, is involved in many physiological and pathological processes. Decorin functions not only as structural molecule in organizing the extracellular matrix but also as signaling molecule controlling cell growth, morphogenesis and immunity. Mutations in decorin or alterations in the post-translational modifications of the glycosaminoglycan (GAG) chain lead to connective tissue disorders such as the congenital stromal corneal dystrophy and the Ehlers-Danlos syndrome. The summarized data reveal that decorin has a large impact on biological processes also because of the complex structure of the GAG chain. The complexity of decorin also covers the binding and sequestering of growth factors and their signaling. This shows that the decorin protein and the dermatan sulfate chain of decorin have both a structural function and a signaling function. Since defects in the biosynthesis of either the protein core or the GAG chain lead to structural alterations in the extracellular matrix and changes in the protein expression profile of the cells embedded in the matrix, this review focuses on the insights of structural function of decorin and includes data about dermatan sulfate.
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Application of High Performance Mass Spectrometry to Structural Analysis of Glycosaminoglycan Oligosaccharides
NATO Science for Peace and Security Series A: Chemistry and Biology, 2008Co-Authors: Daniela G. SeidlerAbstract:Glycosaminoglycans, including those attached to core proteins of proteoglycans, are linear polyanions composed of hexuronate residues alternating with either glucosamine- or galactosamine derivatives. In Galactosaminoglycans the disaccharide subunits can be modified in varying locations within the chains to a variable extent. The modification reaction of sulfation and epimerization lead to specific saccharide sequences controlling a wide range of recently recognized, pivotal functions in tissue development, homeostasis, and repair, and in tumor metastasis. To understand the biological function of Galactosaminoglycans it is important to know their biosynthesis and the fine structure.
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Decorin and its Galactosaminoglycan chain: extracellular regulator of cellular function?
IUBMB life, 2008Co-Authors: Daniela G. Seidler, Rita DreierAbstract:Summary A molecular network of extracellular matrix molecules determines the tissue architecture and accounts for mechanical properties like compressibility or stretch resistance. It is widely accepted that the elements of the cellular microenvironment are important regulators of the cellular behavior in vitro and in vivo. One large group comprising these molecules is the family of proteoglycans. Both, the core proteins and, in particular, the attached Galactosaminoglycans, contribute to the regulation network as they bind a variety of signaling molecules, e.g. cytokines, chemokines, growth, and differentiation factors. We would like to emphasize specific patterns of epimerization and sulfation within the Galactosaminoglycans chains, because these result in ‘‘motifs’’ that are responsible for the modulation of signal factor binding, release and activity. This property is crucial in physiological and pathological conditions, for example development and wound healing. 2008 IUBMB IUBMB Life, 60(11): 729–733, 2008
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Galactosaminoglycan function and oligosaccharide structure determination.
TheScientificWorldJournal, 2007Co-Authors: Daniela G. Seidler, Jasna Peter-katalinić, Alina D. ZamfirAbstract:This review will discuss the importance of sequencing long chondroitin sulfate and dermatan sulfate chains specifically derived from decorin. Decorin is a member of the small leucine-rich repeat proteoglycans and ubiquitously expressed primarily in the skin. Sequence information and diverse function of glycosaminoglycans is further influenced by variable expression through the core protein indicating the importance to analyse glycosaminoglycans from specific proteoglycans.
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core protein dependence of epimerization of glucuronosyl residues in Galactosaminoglycans
Journal of Biological Chemistry, 2002Co-Authors: Daniela G. Seidler, Egon Breuer, Jane K Grandeallen, Vincent C Hascall, Hans KresseAbstract:Chondroitin sulfate and dermatan sulfate proteoglycans are distinguished by differences in their proportion of d-glucuronosyl and l-iduronosyl residues, the latter being formed by chondroitin-glucuronate 5-epimerase during or after glycosaminoglycan chain polymerization. To investigate the influence of the core protein on the extent of epimerization, we expressed chimeric proteins in 293 HEK cells constructed from intact or modified Met(1)-Gln(153) of decorin (DCN), which normally has a single dermatan sulfate chain at Ser(34), in combination with intact or modified Leu(241)-Ser(353) of CSF-1, which has a chondroitin sulfate attachment site at Ser(309). Transfected DCN(M1-Q153), like full-length DCN, contained approximately 20% l-iduronate. Conversely, transfected CSF-1(L241-S353), attached C-terminally on the DCN prepropeptide, contained almost exclusively d-glucuronate. Transfected intact chimeric DCN(M1-Q153)-CSF-1(L241-S353), with two glycosaminoglycan chains, also contained almost exclusively d-glucuronate in chains at both sites, as did chimeras in which alanine was substituted for serine at either of the glycosaminoglycan attachment sites. Nevertheless, undersulfated intact chimeric proteoglycan was an effective substrate for epimerization of glucuronate to iduronate residues when incubated with microsomal proteins and 3'-phosphoadenylylphosphosulfate. C-terminal truncation constructs were prepared from the full-length chimera with an alanine substitution at the CSF-1 glycosaminoglycan attachment site. Transfected truncations retaining the alanine-blocked site contained chains with essentially only glucuronate, whereas those further truncated by 49 or more amino acids and missing the modified attachment site contained chains with approximately 15% iduronate. This 49-amino acid region contains a 7-amino acid motif that appears to be conserved in several chondroitin sulfate proteoglycans. The results are consistent with a model in which the core protein, possibly via this motif, is responsible for routing to subcellular compartments with or without sufficient access to chondroitin-glucuronate 5-epimerase for the addition of chains with or without iduronate residues, respectively.
John R. Couchman - One of the best experts on this subject based on the ideXlab platform.
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perlecan and basement membrane chondroitin sulfate proteoglycan bamacan are two basement membrane chondroitin dermatan sulfate proteoglycans in the engelbreth holm swarm tumor matrix
Journal of Biological Chemistry, 1996Co-Authors: John R. Couchman, Rajesh Kapoor, Meenakshi SthanamAbstract:Abstract The presence of proteoglycans bearing Galactosaminoglycan chains has been reported, but none has been identified previously in the matrix of the Engelbreth-Holm-Swarm tumor, which is a source of several basement membrane components. This tumor matrix contains perlecan, a large, low buoyant density heparan sulfate proteoglycan, widespread in many basement membranes and connective tissues. We now identify two distinct proteoglycan species from this tumor source, which are substituted with Galactosaminoglycans and which show basement membrane localization by immunohistochemistry. One species is perlecan but, in addition to being present as a heparan sulfate proteoglycan, it is also present as a hybrid molecule, with dermatan sulfate chains. A minor population of perlecan apparently lacks heparan sulfate chains totally, and some of this is substituted with chondroitin sulfate. The second species is immunologically related to basement membrane-chondroitin sulfate proteoglycan (BM-CSPG) and bears chondroitin sulfate chains. No BM-CSPG was detectable which was substituted with heparan sulfate chains. A combination of immunological and molecular approaches, including cDNA cloning, showed that perlecan and BM-CSPG are distinct in core protein structure. Both are, however, basement membrane components, although there are tissue-specific differences in their distribution.
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Perlecan and Basement Membrane-Chondroitin Sulfate Proteoglycan (Bamacan) Are Two Basement Membrane Chondroitin/Dermatan Sulfate Proteoglycans in the Engelbreth-Holm-Swarm Tumor Matrix
The Journal of biological chemistry, 1996Co-Authors: John R. Couchman, Rajesh Kapoor, Meenakshi SthanamAbstract:Abstract The presence of proteoglycans bearing Galactosaminoglycan chains has been reported, but none has been identified previously in the matrix of the Engelbreth-Holm-Swarm tumor, which is a source of several basement membrane components. This tumor matrix contains perlecan, a large, low buoyant density heparan sulfate proteoglycan, widespread in many basement membranes and connective tissues. We now identify two distinct proteoglycan species from this tumor source, which are substituted with Galactosaminoglycans and which show basement membrane localization by immunohistochemistry. One species is perlecan but, in addition to being present as a heparan sulfate proteoglycan, it is also present as a hybrid molecule, with dermatan sulfate chains. A minor population of perlecan apparently lacks heparan sulfate chains totally, and some of this is substituted with chondroitin sulfate. The second species is immunologically related to basement membrane-chondroitin sulfate proteoglycan (BM-CSPG) and bears chondroitin sulfate chains. No BM-CSPG was detectable which was substituted with heparan sulfate chains. A combination of immunological and molecular approaches, including cDNA cloning, showed that perlecan and BM-CSPG are distinct in core protein structure. Both are, however, basement membrane components, although there are tissue-specific differences in their distribution.
