Expression of Collagen XVIII and Localization of Its Glycosaminoglycan Attachment Sites

Abstract

Collagen XVIII is the only currently known collagen that carries heparan sulfate glycosaminoglycan side chains. The number and location of the glycosaminoglycan attachment sites in the core protein were determined by eukaryotic expression of full-length chick collagen XVIII and site-directed mutagenesis. Three SerGly consensus sequences carrying glycosaminoglycan side chains were detected in the middle and N-terminal part of the core protein. One of the Ser-Gly consensus sequences carried a heparan sulfate side chain, and the remaining two had mixed chondroitin and heparan sulfate side chains; thus, recombinant collagen XVIII was a hybrid of heparan sulfate and chondroitin proteoglycan. In contrast, collagen XVIII from all chick tissues so far assayed have exclusively heparan sulfate side chains, indicating that the posttranslational modification of proteins expressed in vitro is not entirely identical to the processing that occurs in a living embryo. Incubating the various mutated collagen XVIIIs with retinal basement membranes showed that the heparan sulfate glycosaminoglycan side chains mediate the binding of collagen XVIII to basement membranes.The collagens type XVIII and XV are members of the multiplexins, a collagen subfamily that is characterized by multiple alternating collagen and non-collagenous domains in the protein sequence (1, 2). Collagen XVIII came into the public spotlight by the discovery that the C-terminal peptide of collagen XVIII, named endostatin, has anti-angiogenic and anti-tumor activities (3, 4). The anti-angiogenic activity of the peptide led to the idea that collagen XVIII might be involved in the development of the vascular system. The targeted deletion of collagen XVIII in mice (5) and a naturally occurring mutation in human (6), however, showed that its main function is restricted to the development of the vasculature in the eye but has no function in blood vessel development in other parts of the body. The fact that collagen XVIII is, next to collagen IV, the only collagen that is conserved from Drosophila (7) and Caenorhabditis elegans (8) to humans suggests an important function of the protein in evolution. The deduced amino acid sequences from collagen XVIII cDNA in mouse and human suggested that collagen XVIII is a proteoglycan (1, 9). It was later revealed by investigating the naturally occurring protein from chick embryos (10) that collagen XVIII is a heparan sulfate proteoglycan (HSPG), 1 which is, next to perlecan and agrin, the third extracellular matrix HSPG currently known (10, 11). HSPGs are members of a family of cell surface proteins with polysaccharide side chains characterized by alternating uronic acid and glucosamine units (12, 13). They occur as either integral membrane (14, 15) or as secreted extracellular matrix proteins (12). The polysaccharide chains are connected to the core proteins through serine of the Ser-Gly (SG) consensus sequence whereby the presence of nearby acidic amino acids and the repetition of SGs are factors that promote attachment of glyco...

Specific collagen XVIII isoforms promote adipose tissue accrual via mechanisms determining adipocyte number and affect fat deposition

“…Collagen XVIII has attracted much interest on several accounts. It has the structural properties of both a collagen and a proteoglycan (2); it contains the antiangiogenic molecule, endostatin (3), and its coding gene, collagen α1 (XVIII) (COL18A1), is mutated in the rare, recessively inherited Knobloch syndrome (4). This condition is characterized by high myopia and vitro-retinal degeneration, and occasionally by occipital encephalocele (5,6).…”

Agrin Is a Chimeric Proteoglycan with the Attachment Sites for Heparan Sulfate/Chondroitin Sulfate Located in Two Multiple Serine-Glycine Clusters

“…Finally, clusters 1 and 2 are conserved between the available agrin cDNA sequences from ray, rat, human, and chick. The interspecies conservation of SG consensus sequences turned out to be the best predictor for GAG glycosylation in collagen XVIII (8). We are therefore confident that the only GAG attachment sites of agrin are the ones detected in the two SG clusters.…”

“…In collagen XVIII, however, it was only the first serine in a cluster of 3 SGs that was glycosylated. The second serine in the collagen XVIII SG cluster was only important to direct the glycosylation for HS instead of CS, and the third SG was not important to GAG priming at all (8). An effect of adjacent SGs on the type of GAG glycosylation was not observed for the first multiple SG site (PF9) since the GAG side chains remained the same after mutations of the adjacent serines.…”

“…Better knowledge of the amino acid sequence of the PG glycosylation sites would help to define the rule of glycosylation more clearly. Although the GAG attachment sites of perlecan (3), collagen XVIII (8), and the cell surface HSPGs have been determined (4,9), the sites in agrin are still unknown.…”

New Alternatives for Atherosclerosis Treatment Based on Immunomodulation

“…The aorta is an abundant tissue source of the heparan sulfate proteoglycan (PG) collagen XVIII and its proteolytically released endostatin (ES) fragment, which has previously been shown to inhibit angiogenesis in cancer and atherosclerosis models [37]. The ES portion of collagen XVIII has no attachment sites for GAG but has high affinity for heparin, which is necessary for its antiangiogenesis functions [38]. A recent data has shown that collagen XVIII is differentially degraded in blood vessels affected by atherosclerosis.…”