Sadvakassova Gulzhakhan, Dobocan Monica C, Congote Luis F
Endocrine Laboratory, McGill University Health Centre, 687 avenue des pins, ouest, Montreal, Canada.
BMC Res Notes. 2009 Oct 23;2:215. doi: 10.1186/1756-0500-2-215.
C21, the C-terminal peptide of thrombospondin-4, has growth promoting activity and was discovered as one of several erythropoietin-dependent endothelial proteins. C21 stimulates red cell formation in anemic mice and is a growth factor for CD34+ and CD36+ hematopoietic cells, skin fibroblasts and kidney epithelial cells. ROD1 has been identified as an intracellular mediator. Nothing is known about the existence of putative C21 receptors on plasma membranes of target cells.
We analyzed the nature of C21-binding proteins in cell lysates of skin fibroblasts using C21 affinity columns. The membrane receptor CD44 was identified as C21-binding protein by mass spectrometry. We were unable to demonstrate any direct involvement of CD44 on cell growth or the effect of C21 on cell proliferation. A soluble form of CD44 was synthesized in insect cells and purified from culture supernatants with a combination of PVDF filtration in the presence of ammonium sulphate and HPLC. Both osteopontin and hyaluronic acid competitively displaced Biotin-C21 binding to CD44. In a colony-forming assay using primitive CD133+ hematopoietic stem cells from cord blood, osteopontin and C21 had opposite effects and C21 reduced the inhibitory action of osteopontin.
CD44 is a C21-binding membrane protein. We could not demonstrate an involvement of CD44 in the proliferative action of C21. Nevertheless, based on the antagonism of C21 and osteopontin in hematopoietic precursors, we speculate that C21 could indirectly have a major impact on hematopoietic stem cell proliferation, by hindering osteopontin membrane binding at the level of the bone marrow niche.
血小板反应蛋白-4的C末端肽C21具有促进生长的活性，是作为几种促红细胞生成素依赖性内皮蛋白之一被发现的。C21可刺激贫血小鼠的红细胞生成，是CD34+和CD36+造血细胞、皮肤成纤维细胞和肾上皮细胞的生长因子。ROD1已被确定为一种细胞内介质。关于靶细胞质膜上是否存在假定的C21受体尚不清楚。
我们使用C21亲和柱分析了皮肤成纤维细胞裂解物中C21结合蛋白的性质。通过质谱鉴定膜受体CD44为C21结合蛋白。我们无法证明CD44对细胞生长有任何直接影响，也无法证明C21对细胞增殖的作用。在昆虫细胞中合成了可溶性CD44，并通过在硫酸铵存在下结合PVDF过滤和HPLC从培养上清液中纯化。骨桥蛋白和透明质酸都能竞争性地取代生物素-C21与CD44的结合。在使用来自脐带血的原始CD133+造血干细胞的集落形成试验中，骨桥蛋白和C21具有相反的作用，C21可降低骨桥蛋白的抑制作用。
CD44是一种C21结合膜蛋白。我们无法证明CD44参与了C21的增殖作用。然而，基于C21和骨桥蛋白在造血前体细胞中的拮抗作用，我们推测C21可能通过在骨髓龛水平上阻碍骨桥蛋白与膜的结合，间接对造血干细胞增殖产生重大影响。
