Swann D A, Garg H G, Hendry C J, Hermann H, Siebert E, Sotman S, Stafford W
Department of Surgery, Shriners Burns Institute, Boston, MA 02114.
Coll Relat Res. 1988 Jul;8(4):295-313. doi: 10.1016/s0174-173x(88)80002-5.
Dermatan sulfate (DS) proteoglycans (PGs) were extracted from human post-burn scar (Sc) tissues with 4M guanidinium chloride and isolated from the extracts by DEAE-cellulose chromatography and by differential ethanol precipitation. The DS.PGs were further purified by Sepharose CL-6B column chromatography. The average molecular weight (Mr) of hypertrophic scar (HSc) tissue DS.PGs was 39,000 based on sedimentation equilibrium measurements. Alkaline borohydride treatment of DS.PGs liberated glycosaminoglycan (GAG) chains and the presence of xylitol indicated that these chains were attached to protein core by xylosyl residues. The average Mr of the DS.GAG chain from HSc and normal scar (NSc) samples were 23,500 and 20,000 respectively. After digestion of the HSc and NSc, DS.PGs with chondroitinase ABC in the presence of proteinase inhibitors, two peptide components with Mr values of 21,500 and 17,000 were detected by SDS-polyacrylamide gel electrophoresis using reducing conditions. Analysis of the protein core fractions derived from NSc and HSc DS.PGs by Sepharose CL-6B column chromatography showed the presence of a single NH2-terminal amino acid (aspartic acid) and also that the fractions with different KAV values had an identical NH2-terminal sequence (A1-A5). The A1-A23 sequence of NSc DS.PG (major fraction, C): NH2Asp-Glu-Ala-O-Gly-Ile-Gly-Pro-Glu-Val-Pro-Asp-Asp-Arg-Asp-Phe-G lu-Pro- Ser-Leu-Gly-Pro-Val was the same as reported for a DS.PG isolated from human fetal membrane (HFM) tissue (Brennan et al., 1984). ELISA inhibition assay using monoclonal antibodies raised in rabbit against the NH2-terminal peptide (containing 15 amino acids) of human fetal membrane tissue were found to cross-react with HSc and NSc DS.PGs. Monoclonal antibodies to bovine skin DS.PGs protein core (Pearson et al., 1983) did not show any cross-reactivity with scar DS.PGs. These results show that the scar DS.PGs described here are different from normal bovine skin DS.PGs in the size and type of the protein core, and that in all the samples, the peptide components have the same NH2-terminal amino acid sequence.
用4M氯化胍从人烧伤后瘢痕组织中提取硫酸皮肤素（DS）蛋白聚糖（PGs），并通过DEAE - 纤维素色谱法和分级乙醇沉淀法从提取物中分离出来。DS.PGs通过琼脂糖CL - 6B柱色谱法进一步纯化。基于沉降平衡测量，肥厚性瘢痕（HSc）组织DS.PGs的平均分子量（Mr）为39,000。对DS.PGs进行碱性硼氢化钠处理可释放糖胺聚糖（GAG）链，木糖醇的存在表明这些链通过木糖基残基连接到蛋白核心上。来自HSc和正常瘢痕（NSc）样本的DS.GAG链的平均Mr分别为23,500和20,000。在蛋白酶抑制剂存在的情况下，用软骨素酶ABC消化HSc和NSc的DS.PGs后，在还原条件下通过SDS - 聚丙烯酰胺凝胶电泳检测到两个Mr值分别为21,500和17,000的肽组分。通过琼脂糖CL - 6B柱色谱法对源自NSc和HSc DS.PGs的蛋白核心组分进行分析，结果显示存在单一的NH2末端氨基酸（天冬氨酸），并且具有不同KAV值的组分具有相同的NH2末端序列（A1 - A5）。NSc DS.PG（主要组分，C）的A1 - A23序列：NH2天冬氨酸 - 谷氨酸 - 丙氨酸 - O - 甘氨酸 - 异亮氨酸 - 甘氨酸 - 脯氨酸 - 谷氨酸 - 缬氨酸 - 脯氨酸 - 天冬氨酸 - 天冬氨酸 - 精氨酸 - 天冬氨酸 - 苯丙氨酸 - 谷氨酸 - 脯氨酸 - 丝氨酸 - 亮氨酸 - 甘氨酸 - 脯氨酸 - 缬氨酸与从人胎膜（HFM）组织分离的DS.PG报道的序列相同（Brennan等人，1984）。使用针对人胎膜组织的NH2末端肽（含15个氨基酸）在兔中产生的单克隆抗体进行的ELISA抑制试验发现，该抗体与HSc和NSc DS.PGs发生交叉反应。针对牛皮肤DS.PGs蛋白核心的单克隆抗体（Pearson等人，1983）与瘢痕DS.PGs未显示任何交叉反应。这些结果表明，此处描述的瘢痕DS.PGs在蛋白核心的大小和类型上与正常牛皮肤DS.PGs不同，并且在所有样本中，肽组分具有相同的NH2末端氨基酸序列。
