十字花科黑腐病菌外膜蛋白XpsD與被分泌的澱粉酵素間作用區域的探討

Abstract

XpsD是十字花科黑腐病菌分泌胞外酵素所必須的蛋白質之一。過去的研究指出此蛋白質是以複合體 ( multimer ) 的形式嵌在外胞膜中，且在分泌胞外酵素的過程中，和胞外酵素 (α-Amylase ) 有交互作用 (interaction) 的現象。為進一步了解XpsD和澱粉酵素之間的交互作用，本研究利用免疫沉澱及西方墨點法，首先分析各種N-端或C-端刪除的變異XpsD蛋白與分泌受阻的澱粉酵素間的交互作用，結果發現，具部份 N-domain的XpsD 變異蛋白 ( pHM4 ) 可與澱粉酵素一起被澱粉酵素抗體沉澱，而僅有C-domain的XpsD變異蛋白 ( pCD105 ) 則否，顯示澱粉酵素與XpsD蛋白共同沉澱的現象與其N-domain有關，且只須具備位於氨基酸29-73及304-428兩個區域，即可偵測到免疫共沉澱的訊號。其次，本研究進一步在XpsDD74-303變異蛋白中殖入不同長度的片段，以便分析此區域對澱粉酵素與XpsD蛋白交互作用的影響。結果顯示位於氨基酸123-224及218-303兩個區域的加入，對免疫共沉澱的訊號有加強的效應。最後針對全長的XpsD蛋白、或C端不完整的XpsDD448-650蛋白，在位於氨基酸218-224的區域同樣作三種連續三個氨基酸的刪除、或兩種不同長度氨基酸的插入，並以免疫共沉澱分析，結果顯示澱粉酵素與全長的或C端不完整的XpsD蛋白之免疫共沉澱方式可能不同；且XpsD蛋白氨基酸序列位於218-224區域的刪除變異，對全長XpsD蛋白與澱粉酵素免疫共沉澱訊號強度的影響不顯著；此外，myc 插入全長XpsD蛋白所呈現免疫共沉澱訊號的減弱程度，也不太可能歸因於其與澱粉酵素間專一性識別區域的破壞。由本研究結果推論，XpsD蛋白的N-端在全長蛋白與澱粉酵素的交互作用中所扮演的角色，可能是維持通道的正常整體結構，而非提供局部的專一性識別區。

XpsD is an outer membrane protein that is required for the secretion of extracellular protein in Xanthomonas campestris pv. campestris. Previous studies indicated that the XpsD protein forms a multimer, and it appears to interact with the secreted a-amylase. In this study, we conducted co-immune precipitation followed by immunoblot analysis to characterize the interaction between the XpsD protein and a-amylase. The N-domain truncated XpsDD29-428 (pCD105) protein was not precipitated by antibody against a-amylase, whereas addition of amino acid residues 29-73 and 304-428 (XpsDD74-303, pHM4) was sufficient to observe co-immune precipitation between the two proteins. These results suggested that interaction between the XpsD protein and a-amylase is probably through the N-domain of the XpsD. Subsequent attempt to investigate the significance in the region missing in XpsDD74-303 revealed a short region within amino acid residues 218-224 that was shared by two truncated XpsD proteins and exhibited enhanced co-immune precipitation. To investigate the possible involvement of this region in specific recognition between the XpsD and a-amylase, I constructed three 3-amino acid deletions and two insertions in the full-length XpsD and the C-domain truncated XpsDD448-650 and conducted further co-immune precipitation analysis. The results indicated that co-immune precipitation between the full-length XpsD and the C-domain truncated XpsDD448-650 with a-amylase are probably through different modes of interaction. Furthermore, deletions within amino acid residues 218-224 did not cause significant effect upon co-immune precipitation between the full-length XpsD and a-amylase. Reduction of co-immune precipitation between the full-length XpsD and a-amylase caused by insertion of the myc epitope downstream of the amino acid residue 222 was not great enough to suggest a specific recognition region located within this region. None of the results from this study is supportive of a specific recognition region in the N-domain of the XpsD protein in the interaction between XpsD and a-amylase. Perhaps the N-domain of the XpsD protein is required for maintaining the correct overall structure to allow passage of a-amylase through the channel.