Voigt J, Liebich I, Hinkelmann B, Kiess M
Botanisches Institut, Technische Universität Braunschweig, Germany.
Plant Cell Physiol. 1996 Jan;37(1):91-101. doi: 10.1093/oxfordjournals.pcp.a028919.
To identify precursors of the insoluble glycoprotein framework of the Chlamydomonas cell wall, a polyclonal antibody was raised against the mixture of polypeptides released from the insoluble wall fraction by chemical deglycosylation. This antibody preferentially cross-reacted with a '150 kDa' salt-soluble cell wall glycoprotein. The conclusion that this '150 kDa' glycoprotein is a putative precursor of the insoluble cell wall fraction was corroborated by the results of pulse-chase experiments and by experiments with antibodies raised against the '150 kDa' salt-soluble glycoprotein and against its 100 kDa deglycosylation product, respectively. Whereas the antibody against the '150 kDa' glycoprotein preferentially recognized carbohydrate side chains, the antibody against its 100 kDa deglycosylation product was found to have essentially the same specificity towards glycosylated and deglycosylated cell wall components as the antibody against the deglycosylation products of the insoluble wall fraction. Furthermore, the antibody against the deglycosylated, insoluble wall fraction recognized almost the same set of peptide fragments derived by V8 protease treatment from the '150 kDa' salt-soluble cell wall glycoprotein and its 100 kDa deglycosylation product, respectively, as the antibody against the 100 kDa deglycosylated cell wall polypeptide.
为了鉴定衣藻细胞壁不溶性糖蛋白骨架的前体，制备了一种多克隆抗体，该抗体针对通过化学去糖基化从不溶性细胞壁组分中释放的多肽混合物产生。这种抗体优先与一种“150 kDa”的盐溶性细胞壁糖蛋白发生交叉反应。脉冲追踪实验以及分别用针对“150 kDa”盐溶性糖蛋白及其100 kDa去糖基化产物产生的抗体进行的实验结果，证实了这种“150 kDa”糖蛋白是不溶性细胞壁组分的假定前体这一结论。针对“150 kDa”糖蛋白的抗体优先识别碳水化合物侧链，而针对其100 kDa去糖基化产物的抗体，被发现对糖基化和去糖基化的细胞壁组分具有与针对不溶性细胞壁组分去糖基化产物的抗体基本相同的特异性。此外，针对去糖基化的不溶性细胞壁组分的抗体，分别从“150 kDa”盐溶性细胞壁糖蛋白及其100 kDa去糖基化产物中识别出几乎相同的一组由V8蛋白酶处理产生的肽片段，这与针对100 kDa去糖基化细胞壁多肽的抗体相同。
