Molecular cloning and characterization of Ac-TMP-2, a tissue inhibitor of metalloproteinase secreted by adult Ancylostoma caninum.
Ac-TMP-2, an immunodominant hookworm antigen encoding a tissue inhibitor of metalloproteinase (TIMP) was cloned by immunoscreening an Ancylostoma caninum larval cDNA library with sera pooled from dogs immunized with irradiated A. caninum third stage larvae (ir-L3). The open reading frame of Ac-tmp-2 cDNA encoded a 244 amino acids (predicted molecular weight of 27.7 kDa), which shared a common N-terminus with other vertebrate and invertebrate TIMPs, including Ac-TMP-1, the most abundant adult hookworm secreted protein. However Ac-TMP-2 also contains an unusual multicopy (ten) repeat of the amino acid sequence, KTVEENDE. By immunoblotting, Ac-TMP-2 was detected only in adult hookworms and their excretory secretory products although the corresponding mRNA was also detected in L3. Immunolocalization with specific antiserum showed that native Ac-TMP-2 was located in adult worm's esophagus and cephalic glands. Recombinant Ac-TMP-2 expressed in bacteria was highly immunogenic and recognized by ir-L3 immunized dog immune sera. The recombinant Ac-TMP-2 protein inhibited the human matrix metalloproteinases, MMP-2, MMP-7 and MMP-13. As an immunodominant protein having a possible role in the parasite-host relationship of canine hookworm infection, recombinant Ac-TMP-2 represents a plausible target for vaccine development.
Zhan
Bin
B
Department of Microbiology, Immunology and Tropical Medicine, The George Washington University and the Sabin Vaccine Institute, Washington, DC 20037, USA. mtmbxz@gwumc.edu
Gupta
Richi
R
Wong
Susan P Y
SP
Bier
Stacia
S
Jiang
Desheng
D
Goud
Gaddam
G
Hotez
Peter
P
D000595:Amino Acid Sequence; D000722:Ancylostoma / Q000201:enzymology / Q000378:metabolism*; D000818:Animals; D000947:Antigens, Helminth / Q000032:analysis / Q000737:chemistry* / Q000235:genetics*; D003001:Cloning, Molecular; D004285:Dogs; D015801:Helminth Proteins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics*; D006801:Humans; D053505:Matrix Metalloproteinases, Secreted / Q000037:antagonists & inhibitors / Q000378:metabolism; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019714:Tissue Inhibitor of Metalloproteinases / Q000032:analysis / Q000737:chemistry* / Q000235:genetics*
D000947:Antigens, Helminth; D015801:Helminth Proteins; D019714:Tissue Inhibitor of Metalloproteinases; D053505:Matrix Metalloproteinases, Secreted