Molecular biological study on the hemagglutinin with protcolytic activity (HA/P) in Porphyromonas gingivalis

牙龈卟啉单胞菌（P.g.）是一种革兰氏阴性的厌氧杆菌，被认为是成人牙周炎的潜在病原体。P.G.具有活性被精氨酸、蛋白酶抑制剂如TLCK和亮抑酶肽抑制的非凝集素样血凝素（HA）。HA还具有半胱氨酸蛋白酶活性（P），其切断精氨酸残基旁边的键。这种双功能因子被称为HA/P，据报道由单一种类的蛋白质组成，其分子量为44 kDa。HA/P最近被认为是主要的组织破坏剂之一。为了进一步了解HA和P之间的结构关系及其调控机制，我们克隆了HA/P的结构基因hap，并将其重组到大肠杆菌中进行表达。i）hap基因长度大于5 kbp。我们成功地克隆了一个4kbp长的DNA片段。该片段仅含18个氨基酸的N-末端氨基酸残基， ...更多信息 用反向PCR法进一步克隆了hap基因的下游序列。orf持续超过2kbp，没有结束。对HA/P基因上游区进行核苷酸序列测定，发现其ORF位于HA/P N端2.3kbp处。Pavloff等克隆了HA/P的全基因，其序列与我们报道的几乎全部序列一致。ii）HA/P是复合物。iii）HA/P复合物的亚基在一个连续的ORF中编码。HA/P被认为是通过自催化作用后熟的。iv）称为Arg-牙龈菌蛋白酶的半胱氨酸蛋白酶与其前导肽和前肽的序列一起在上游区域编码，即，Arg-牙龈菌蛋白酶在前酶原中翻译。v）HA被认为是在下游区域编码的，尽管没有直接证据。vi）我们成功地在大肠杆菌中表达了插入到pT 7表达载体中的hap的N-末端区域。表达肽的N端氨基酸序列为QTTEL等，因此不能通过实验确定起始密码子。（vii）以hap基因的p_s为探针的Southern杂交实验表明，与抗HA/P（P.g.）牙髓卟啉单胞菌hap抗体的核苷酸序列与牙髓卟啉单胞菌hap的核苷酸序列同源性较低。少

Porphyromonas gingivalis (P.g.) is a gram-negative, anacrobic rod, which is believed to be a potential pathogen for an adult periodontitis. P.g. has a non-lectin-like hemagglutinin (HA) of which the activity is inhibited by arginine, protease inhibitors such as TLCK and leupeptin. The HA also has a cysteine protease activity (P) which cuts the bond next to an arginine residue. This bifunctional factor, called HA/P,was reported to be composed of a single kind of protein, the molecular weight of which is 44 kDa. HA/P has recently been thought to be one of the major tissue destructive agents. To help understand the structural relaitonship between HA and P and the mechanism for the regulation of these functions, we attempted to clone the structural gene of HA/P,named hap, recombine it into an expression vector, and express it in Escherichia coli. i) hap gene is more than 5 kbp long. We succeeded in cloning a 4 kbp-long DNA fragment. This fragment contained only 18 amino acid N-terminal por … More tion of HA/P.The downstream region of hap was further cloned by inverse PCR method. The orf continued more than 2 kbp and did not end. The nucleotide scquencing of the upstream region revealed the orf continuting about 2.3 kbp from the N-terminus of HA/P.The whole gene of hap was cloned by Pavloff et al. The sequence coincided with almost all of ours. ii) HA/P is a complex. iii) The subunits of the HA/P complex are encoded in one continuous orf. HA/P is thought to be matured by autocatalysis posttranslationally. iv) A cysteine protease called Arg-gingipain is encoded in the upstream region together with the sequences for its leader peptide and propeptide, that is, Arg-gingipain is translated in preproenzyme. v) HA is thought to be encoded in the downstream region, although there was no direct evidence. vi) We succeeded in expressing the N-terminal region of hap inserted in the pT7 expression vector in E.coli. The N-terminal amino acid sequence of the expressed peptide is QQTEL etc. Thus the initiation codon could not be determined experimentally. vii) Southern hybridization experiment with p ; arts of hap gene as a probe indicated that a material crossreacting with anti-HA/P (P.g.) antibody in P.endodontalis has a nucleotide sequence with low homology with that of hap in P.g. Less