Laskin D L, Kimura T, Sakakibara S, Riley D J, Berg R A
J Leukoc Biol. 1986 Mar;39(3):255-66. doi: 10.1002/jlb.39.3.255.
Damage to interstitial connective tissue is associated with the rapid accumulation of monocytes and neutrophils at the site of injury. To study the role of collagen fragments in neutrophil migration, we analyzed the chemotactic properties of peptide fragments of bovine collagen digested with bacterial collagenase or cyanogen bromide and small molecular weight synthetic polypeptides containing proline (Pro), hydroxyproline (Hyp), and glycine (Gly), the major amino acids that comprise collagen. Using the Boyden chamber and under agarose techniques, we found that collagen fragments were as potent in inducing chemotaxis in neutrophils as the bacterial-derived peptide f-met-leu-phe. The synthetic polytripeptides (Pro-Pro-Gly)5 and (Pro-Hyp-Gly)5 were found to be equipotent in inducing chemotaxis, producing a maximal induction of chemotaxis at 5-10 nM. This suggests that Hyp, the unique imino acid found in collagen, is not required for chemotactic activity. Increasing the length of the synthetic tripeptide from 5 to 10 subunits decreased its chemotactic activity, while the single tripeptide subunit (Pro-Hyp-Gly)1 was the least active peptide, inducing a maximal response at 100 nM. To study the structural requirements for chemotaxis, Pro-Hyp-Gly tripeptides were synthesized with modifications at the N and C terminals ends. Addition of a methyl group to the carboxyl of Gly to form an ester enhanced the chemotactic activity of the peptide by 50%, while substitutions on the amino terminus with an acetyl group decreased the chemotactic activity by 50%. Substitution on the amino terminus with a Boc group decreased the chemotactic activity by 100%. These results indicate that there are specific structural requirements for chemotaxis induced by peptides having a collagen-like sequence of amino acids.
间质结缔组织损伤与单核细胞和中性粒细胞在损伤部位的快速聚集有关。为了研究胶原蛋白片段在中性粒细胞迁移中的作用，我们分析了用细菌胶原酶或溴化氰消化的牛胶原蛋白肽片段以及含有脯氨酸（Pro）、羟脯氨酸（Hyp）和甘氨酸（Gly）（构成胶原蛋白的主要氨基酸）的小分子合成多肽的趋化特性。使用博伊登小室和琼脂糖技术，我们发现胶原蛋白片段在诱导中性粒细胞趋化方面与细菌衍生的肽f - met - leu - phe一样有效。合成的聚三肽（Pro - Pro - Gly）5和（Pro - Hyp - Gly）5在诱导趋化方面具有同等效力，在5 - 10 nM时产生最大趋化诱导。这表明胶原蛋白中独特的亚氨基酸Hyp对于趋化活性并非必需。将合成三肽的长度从5个亚基增加到10个亚基会降低其趋化活性，而单个三肽亚基（Pro - Hyp - Gly）1是活性最低的肽，在100 nM时诱导最大反应。为了研究趋化的结构要求，合成了在N端和C端有修饰的Pro - Hyp - Gly三肽。在Gly的羧基上添加一个甲基形成酯可使肽的趋化活性提高50%，而在氨基末端用乙酰基取代会使趋化活性降低50%。在氨基末端用Boc基团取代会使趋化活性降低100%。这些结果表明，具有胶原蛋白样氨基酸序列的肽诱导趋化存在特定的结构要求。
