Localization of a vitamin‐D‐binding protein interaction site in the COOH‐terminal sequence of actin

Abstract

The serum vitamin D binding protein is the carrier of vitamin D and its derivatives in the plasma. One of the known roles of this protein is to sequester monomeric actin in the blood, therefore implicating this protein in actin elimination. However, its binding site at the surface of actin is poorly delimited. We report here the results of a study which locates, using several actin fragments together with immunological probes, a vitamin D binding protein site near the COOH-terminal extremity.Thus, the interface is delimited by the sequence 360-372 in subdomain I of actin.The vitamin D binding protein (DBP), also called Gc (group component), is a plasma protein found in humans and other mammalian species [l]. It is a monomeric protein with an a2-globulin mobility, secreted by the liver [2, 31 and has a relative molecular mass (M,) of 52000 [3]. The first recognized function of DBP was the plasma transport of vitamin D and other related derivatives [4]. In addition, other previous studies [5] have shown that the serum affords actin-depolymerizing activity. This property was further ascribed to the conjugate effects of gelsolin and DBP [6]. However, whereas gelsolin can cause severing of actin filaments, DBP can sequester actin monomers in a 1 : l complex and thereby leads to actin depolymerization. The in vivo occurrence of a DBP-actin complex was detectcd, at a very low level, in the serum of healthy patients [7]. A drastic increase in the amount of actin and its DBP complex following tissue injury (hepatic necrosis for example) or trauma have also been reported [7, 81. Thus, one of the physiological roles of DBP would be to avoid actin filament formation, which would occur at normal salinity of the serum, and thus accelerate clearing of actin from the circulation [9, 101.In vitru, studies have clearly shown the existence of a very tight G-actin -DBP complex without interaction with F-actin [6]. Other studies have also suggested that the interaction site is restricted to the 227-375 segment of actin [ll], the Cterminal extremity being excluded. Another actin-sequestering protein, profilin, binds with a relatively low affinity at or near the C-terminal extremity of actin [12]. Thus, the two proteins would have different binding sites on actin. In fact, other actin-associated proteins have been shown to be directed to the C-terminal part of the actin sequence. This is true, not only for proteins which prevent actin polymerization, but also for crosslinking proteins such as a-actinin [13], for severing proteins such as gelsolin which interact with the 305 -326 actin sequence [14] and for the myosin subfragment-1 (SI)In view of the potential importance of DBP in the elimination of actin from plasma, we have begun a study aimed at defining the location of the DBP interface in the actin sequence. We located an interaction site of DBP precisely in subdomain I of actin. ~5 1 . MATERIALS AND METHODS Materials7-Chloro-4-nitrobenzo-2-oxa-I ,3,diazole (Nbd-C1) and Niodoacetyl-N-(5-sulfo-l-naphthyl) ethylenediamine (1,5-I-AED...

Toward a Human Blood Serum Proteome

“…Examples of unexpected results are hemoglobin and actin, which are both ubiquitous in the red blood cells. Therefore between high quantity and rapid turnover of red blood cells it may be expected that hemoglobin and actin should be readily detectable in serum (42). In contrast to our expectations, few hemoglobin-derived peptides and no actin-derived peptides were identified.…”

“…In contrast to our expectations, few hemoglobin-derived peptides and no actin-derived peptides were identified. In fact, both hemoglobin and actin are actively sequestered and cleared from the serum via the abundant serum proteins haptoglobin and vitamin D-binding protein, respectively (42)(43)(44)(45). Another example of unexpected results are the identification of immunoglobulin-derived peptides, although depletion was complete when evaluated by SDS-PAGE.…”

Definition of the EGTA-independent interface involved in the serum gelsolin-actin complex

“…This highly sensitive method, which involved immunological and enzymic amplifications, was previously developed to describe interfaces between actin and a-actinin (Lebart et al, 1990), filamin (Mejean et al, 1992), myosin-head (Labbe et al, 1992), vitamin D-binding protein and arginine kinase (Reddy et al, 1992). In addition, the results obtained here were substantiated by fluorescence anisotropy determinations in solution as reported in previous papers (Reddy et al, 1992;Houmeida et al, 1992). In the presence of Ca2+, it is well known that the gelsolin molecule interacts with at least 2 mol of actin.…”

Identification of low abundance cyclophilins in human plasma

“…We found only 15 non‐PPIase proteins, which are usually attributed to the intracellular space. Among these intracellular proteins are hemoglobin and actin known to occur in plasma due to red blood cell lysis .…”