no M, Raab G, Lau K, Abraham J A, Klagsbrun M
Department of Surgery, Children's Hospital, Boston, Massachusetts 02115.
J Biol Chem. 1994 Dec 9;269(49):31315-21.
Heparin-binding epidermal growth factor-like growth factor (HB-EGF), whose cDNA has a predicted 208-codon open reading frame, is synthesized as a membrane-spanning precursor that is processed to release mature mitogenic proteins of approximately 73-87 amino acids in length. Previous work has focused on the structural and biological properties of secreted HB-EGF. In this study, human recombinant transmembrane HB-EGF, produced by expression of HB-EGF1-208 cDNA in a baculovirus system, has been isolated, purified, and characterized structurally and biologically. Two isoforms of transmembrane HB-EGF (HB-EGFTM) were purified from membrane fractions of infected insect cells by a combination of heparin affinity chromatography and reversed-phase high performance liquid chromatography. The isoform designated as HB-EGFTM-1, a 21.5-kDa protein, yielded no N-terminal sequence, suggesting that it is N-terminally blocked. However, HB-EGFTM-II, a 24-kDa protein, was N-terminally sequenced and found to be initiated at Asp63 in the 208-amino acid residue primary translation product. This N terminus is the same as that determined for a 18-kDa isoform of secreted HB-EGF purified from the conditioned medium of insect cells expressing HB-EGF1-149 cDNA and is also identical to the N terminus of the longest form of secreted HB-EGF initially purified from human macrophage-like U-937 cell conditioned medium. HB-EGFTM-II cross-reacted on a Western blot with an antibody directed against the 16 C-terminal amino acids of the cytoplasmic tail of HB-EGF, indicating that it contains a putative transmembrane domain. HB-EGFTM-II was bioactive and stimulated the proliferation of BALB/c 3T3 cells and smooth muscle cells and the motility of smooth muscle cells, albeit with approximately 10-25% of the specific activity of secreted HB-EGF isoforms. We concluded that transmembrane HB-EGF is bioactive when isolated, consistent with the possibility of its functioning as a juxtacrine growth factor when still tethered to the cell.
肝素结合表皮生长因子样生长因子（HB-EGF），其cDNA具有一个预测的208个密码子的开放阅读框，是以跨膜前体形式合成的，该前体经过加工后释放出长度约为73-87个氨基酸的成熟促有丝分裂蛋白。以往的研究主要集中在分泌型HB-EGF的结构和生物学特性上。在本研究中，通过在杆状病毒系统中表达HB-EGF1-208 cDNA产生的人重组跨膜HB-EGF已被分离、纯化，并进行了结构和生物学特性鉴定。通过肝素亲和色谱和反相高效液相色谱相结合的方法，从感染昆虫细胞的膜组分中纯化出两种跨膜HB-EGF（HB-EGFTM）同工型。命名为HB-EGFTM-1的同工型是一种21.5 kDa的蛋白质，未得到N端序列，表明其N端被封闭。然而，24 kDa的蛋白质HB-EGFTM-II经N端测序后发现，它在208个氨基酸残基的初级翻译产物中从Asp63起始。这个N端与从表达HB-EGF1-149 cDNA的昆虫细胞条件培养基中纯化的18 kDa分泌型HB-EGF同工型所确定的N端相同，也与最初从人巨噬细胞样U-937细胞条件培养基中纯化的最长形式的分泌型HB-EGF的N端相同。HB-EGFTM-II在蛋白质免疫印迹中与针对HB-EGF细胞质尾16个C端氨基酸的抗体发生交叉反应，表明它含有一个假定的跨膜结构域。HB-EGFTM-II具有生物活性，能刺激BALB/c 3T3细胞和平滑肌细胞的增殖以及平滑肌细胞的运动，尽管其比活性约为分泌型HB-EGF同工型的10%-25%。我们得出结论，跨膜HB-EGF分离后具有生物活性，这与其在仍与细胞相连时作为旁分泌生长因子发挥作用的可能性一致。
