Tumor shedding of laminin binding protein modulates angiostatin production in vitro and interferes with plasmin-derived inhibition of angiogenesis in aortic ring cultures.
The growth of solid tumors is largely controlled by the process of angiogenesis. A 67 kDa protein, the laminin binding protein (LBP), is shed from malignant cells in significant amounts and binds to laminin-1 (Starkey et al., Cytometry 1999;35:37-47; Karpatová et al., J Cell Biochem 1996;60:226-34). However, the functions of shed LBP are not fully understood. We hypothesize that matrix-bound LBP could modulate local tumor angiogenesis. In support of this hypothesis, we demonstrate that shed LBP exhibits sulfhydryl oxidase-like activities, and modifies the production of angiostatins from plasmin in vitro. The molecular weights of the autocatalytic products of lys-plasmin incubated with LBP in vitro suggest that PMDs (plasmin A chains attached to degraded B chains) (Ohyama et al., Eur J Biochem 2004;271:809-20) are preferentially generated. Using rat aortic ring assays, we also show that shed LBP reverses plasmin-dependent inhibition of vascular outgrowth. To elucidate which LBP region(s) are active in modulating angiogenesis, limited proteolysis experiments were conducted to determine stable rLBP domains likely to fold correctly, and these were cloned, expressed and purified. The stable LBP fragments were tested for binding to laminin-1 and for competition with shed LBP activity in the aortic ring assay. Results of these studies suggest that the active LBP domains lie within the 137-230 amino acid sequence, a region known to contain 2 bioactive sequences. Since this fragment binds to laminin-1 and modulates angiogenesis, it appears likely that binding of shed LBP to matrix laminin-1 is related to its functions in tumor angiogenesis. The findings presented in this manuscript suggest that LBP shedding could provide a useful therapeutic target.
Moss
Britney L
BL
Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717, USA.
Taubner
Lara
L
Sample
Yekaterina K
YK
Kazmin
Dmitri A
DA
Copié
Valérie
V
Starkey
Jean R
JR
D000818:Animals; D001011:Aorta; D005341:Fibrinolysin / Q000502:physiology*; D007797:Laminin / Q000378:metabolism*; D009369:Neoplasms / Q000098:blood supply / Q000503:physiopathology; D009389:Neovascularization, Pathologic; D011485:Protein Binding; D051381:Rats; D017474:Receptors, Laminin / Q000502:physiology*
D007797:Laminin; D017474:Receptors, Laminin; C121346:laminin 1; C510326:laminin binding protein 67-kDa, rat; D005341:Fibrinolysin