Stoddart J H, Jasuja R R, Sikorski M A, von Andrian U H, Mier J W
Division of Hematology-Oncology and Tupper Research Institute, Department of Medicine, New England Medical Center, Tufts University School of Medicine, Boston, MA 02111, USA.
J Immunol. 1996 Dec 15;157(12):5653-9.
The adhesion molecule L-selectin (CD62L) is rapidly shed from the plasma membrane during leukocyte activation as a result of proteolytic cleavage between Lys321 and Ser322 within the extracellular domain. L-selectin is also down-modulated from the surface in response to cross-linking, possibly through a similar mechanism. To further characterize the mechanism of down-modulation, several L-selectin mutants were generated and transfected into COS cells. Wild-type L-selectin as well as mutants with one or two amino acid substitutions at the cleavage site were nearly quantitatively shed into the culture supernatant. However, mutants in which a nine-amino acid stretch that included the protease-sensitive site was either deleted or replaced with a polyglycine spacer or a comparable region of E-selectin were retained on the cell surface and not detected in the supernatant. These results are consistent with other reports describing protease resistant L-selectin mutants. We also demonstrate that when expressed in L1-2 pre-B cells, the L-selectin nine-amino acid deletion mutant (321del.9), but not wild-type L-selectin, is resistant to down-regulation induced by PMA. However, both wild-type and mutant 321del.9 are completely lost from the cell surface in response to cross-linking with an L-selectin Ab. PMA-induced- but not L-selectin cross-linking-induced down-modulation was inhibited by staurosporine. These data are consistent with the idea that the L-selectin protease(s) can tolerate minor structural alterations at the cleavage site, and that L-selectin down-modulation can be induced by more than one mechanism, at least one of which (cross-linking) is protein kinase C independent.
黏附分子L-选择素（CD62L）在白细胞激活过程中，由于细胞外结构域中赖氨酸321和丝氨酸322之间的蛋白水解切割，会迅速从质膜上脱落。L-选择素也会因交联作用而从表面下调，可能是通过类似的机制。为了进一步表征下调机制，构建了几个L-选择素突变体并转染到COS细胞中。野生型L-选择素以及在切割位点有一个或两个氨基酸替换的突变体几乎定量地脱落到培养上清液中。然而，包含蛋白酶敏感位点的九个氨基酸片段被删除或被聚甘氨酸间隔区或E-选择素的可比区域取代的突变体保留在细胞表面，在上清液中未检测到。这些结果与其他描述蛋白酶抗性L-选择素突变体的报告一致。我们还证明，当在L1-2前B细胞中表达时，L-选择素九个氨基酸缺失突变体（321del.9），而不是野生型L-选择素，对佛波酯（PMA）诱导的下调具有抗性。然而，野生型和突变体321del.9在与L-选择素抗体交联后都会从细胞表面完全消失。星形孢菌素抑制PMA诱导的下调，但不抑制L-选择素交联诱导的下调。这些数据与以下观点一致，即L-选择素蛋白酶能够耐受切割位点的微小结构改变，并且L-选择素的下调可以由多种机制诱导，其中至少一种机制（交联）不依赖蛋白激酶C。
