Baeza M L, Reddigari S R, Kornfeld D, Ramani N, Smith E M, Hossler P A, Fischer T, Castor C W, Gorevic P G, Kaplan A P
Department of Medicine, State University of New York, Stony Brook 11794-8161.
J Clin Invest. 1990 May;85(5):1516-21. doi: 10.1172/JCI114598.
We have previously reported purification of three forms of histamine-releasing factors (HRFs) from mixtures of streptokinase-streptodornase stimulated human mononuclear cells and platelets with apparent molecular masses of 10-12, 15-17, and 40-41 kD (1989. J. Clin. Invest. 83:1204-1210). We have also prepared mouse MAbs against the 10-12-kD HRF (1989. J. Allergy Clin. Immunol. 83:281). Affinity-purified 10-12-kD HRF appears as a broad band upon polyacrylamide gel electrophoresis in the presence of SDS. We determined the NH2-terminal amino acid sequence of the top and bottom halves of this broad band. Sequence analysis revealed striking homology between this HRF and connective tissue activating peptide-III (CTAP-III), a platelet-derived 8-10-kD protein known to cause mitogenesis and extracellular matrix formation in fibroblast cultures. 19 of 21 NH2-terminal residues in the top half of the HRF band were identical to the NH2-terminal sequence of CTAP-III. 20 of 21 NH2-terminal residues in the bottom half were identical to the NH2-terminal sequence of neutrophil-activating peptide-2, which is derived from CTAP-III by proteolytic cleavage between residues 15 and 16. Purified CTAP-III also released histamine from basophils. Rabbit antiserum raised against either native or recombinant CTAP-III recognized affinity-purified HRF in immunodot blot assays, and MAb against HRF recognized CTAP-III in both dot blot and microtiter plate based immunoassays. These data demonstrate the first structural, functional, and immunologic relationship between one form of human HRF and a previously described cell product.
我们之前报道过，从链激酶-链道酶刺激的人单核细胞和血小板混合物中纯化出三种组胺释放因子（HRFs），其表观分子量分别为10 - 12、15 - 17和40 - 41 kD（1989年。《临床研究杂志》83:1204 - 1210）。我们还制备了针对10 - 12-kD HRF的小鼠单克隆抗体（1989年。《变态反应与临床免疫学杂志》83:281）。在SDS存在的情况下，经亲和纯化的10 - 12-kD HRF在聚丙烯酰胺凝胶电泳中呈现为一条宽带。我们测定了这条宽带上下两半部分的NH2末端氨基酸序列。序列分析显示，这种HRF与结缔组织激活肽III（CTAP-III）之间存在显著的同源性，CTAP-III是一种血小板衍生的8 - 10-kD蛋白质，已知可在成纤维细胞培养物中引起有丝分裂和细胞外基质形成。HRF带的上半部分21个NH2末端残基中有19个与CTAP-III的NH2末端序列相同。下半部分21个NH2末端残基中有20个与中性粒细胞激活肽-2的NH2末端序列相同，中性粒细胞激活肽-2是通过CTAP-III在第15和16位残基之间的蛋白水解切割产生的。纯化的CTAP-III也能从嗜碱性粒细胞中释放组胺。针对天然或重组CTAP-III产生的兔抗血清在免疫斑点印迹试验中识别经亲和纯化的HRF，而针对HRF的单克隆抗体在斑点印迹和基于微量滴定板的免疫试验中都能识别CTAP-III。这些数据证明了一种形式的人HRF与一种先前描述的细胞产物之间的首次结构、功能和免疫学关系。
