Monoclonal Antibodies to the Cell-Wall-Associated Proteinase of Lactococcus lactis subsp. cremoris Wg2

Abstract

Twelve monoclonal antibodies directed to the cell-wall-associated proteinase of Lactococcus lactis subsp. cremoris Wg2 were isolated after immunization of BALB/c mice with a partially purified preparation of the proteinase. The monoclonal antibodies reacted with the 126-kilodalton proteinase band in a Western immunoblot. All but one of the monoclonal antibodies reacted with protein bands with a molecular weight below 126,000, possibly degradation products of the proteinase. The monoclonal antibodies could be divided into six groups according to their different reactions with the proteinase degradation products in the Western blot. Different groups of monoclonal antibodies reacted with different components of the L. lactis subsp. cremoris Wg2 proteinase. Crossed immunoelectrophoresis showed that monoclonal antibody groups I, II, and III react with proteinase component A and that groups IV, V, and VI react with proteinase component B. The isolated monoclonal antibodies cross-reacted with the proteinases of other L. lactis subspecies. Monoclonal antibodies of group IV cross-reacted with proteinase component C of other L. lactis subsp. cremoris strains. The molecular weight of the proteinase attached to the cells of L. lactis subsp. cremoris Wg2 was 200,000, which is different from the previously reported values. This could be analyzed by immunodetection of the proteinase on a Western blot. This value corresponds to the molecular weight calculated from the amino acid sequence of the cloned L. lactis subsp. cremoris Wg2 proteinase gene.

The physiology and biochemistry of the proteolytic system in lactic acid bacteria

“…weakening of proteinase-cell wall interactions) exposing other autoproteolytic sites even closer to the Cterminus. Since the M r of the 180-kDa form is close to that predicted for the entire mature enzyme [32,33,41,42], this form must arise from cleavage at a site close to the membrane anchor. Cell lysis is minimal (less than 1%) using the lysozyme plus Ca 2÷ release procedure [55] so it is unlikely that the 180-kDa form includes the membrane anchor region itself.…”

“…If the lactococcal proteinase does have a similar organization to the M6 protein in the wall-associated region this would mean that a segment of approximately 15 kDa at the C-terminus is located within the wall. Since the size of the mature lactococcal proteinase after removal of the Nterminal prepro-sequence is thought to be 180 kDa [32,33,41,42], this should leave the remaining 165 kDa exposed on the outer surface of the cell. The largest fragment reported to be released following incubation in Ca2+-free ' buffer is 165 kDa [41,43] which is consistent with this interpretation.…”

“…Since the size of the mature lactococcal proteinase after removal of the Nterminal prepro-sequence is thought to be 180 kDa [32,33,41,42], this should leave the remaining 165 kDa exposed on the outer surface of the cell. The largest fragment reported to be released following incubation in Ca2+-free ' buffer is 165 kDa [41,43] which is consistent with this interpretation. However, this picture of a homogeneously organized population of proteinase molecules exposed at the outer surface of the cell may be an oversimplification which does not fully account for the kinetics of proteinase release (see below).…”

“…Following release, the proteinase is often found to exist in different molecular mass or antigenically distinct forms [41,50,52,54,55]. It is now clearly established that the different molecular mass forms from any one strain arise by autoproteolysis [54] and are thus artefacts of the release process rather than genetically distinct proteinase types.…”

Mode of action of LciA, the lactococcin A immunity protein

“…ELISA was performed as described previously (Laan et al, 1988). Plates were scanned in a Titertek multiscan (Molecular Devices Corporation) at 490 nm.…”