Hydroxyproline-Rich Bacterial Agglutinin from Potato

Abstract

A protein, extracted from Katahdin potato (Solanum tuberosum L. cv 'Katahdin') tubers and purified by ion exchange chromatography and gel filtration, agglutinates avirulent strains of the bacterial wilt pathogen, Pseudomonas solanacearum, but only weakly agglutinates vinldent strains.The agglutinin has very low hemagglutinating activity (in contrast to potato lectin) and is a glycoprotein containing about 61% carbohydrate. The carbohydrate moiety contains 91% (weight %) arabinose, 5% galactose, 3% glucose, and 1% glucosamine. The protein portion is rich in hydroxyproline (42%), lysine (16%), serine (9%), and proline (9%). The entire agglutinin has a molecular weight of 91,000 ± 5,000 and is very basic (pI > 11). Shape estimations based on the concentration dependence of the sedimentation coefficient, the high viscosity (Qlj = 92.7), the frictional coefficient (f/f0 = 2.15), and axial ratio (a/b = 25) indicate that the agglutinin is a prolate ellipsoid.Proteins that agglutinate bacteria have been extracted from many different plants (30). It has been suggested that these agglutinins are responsible for the attachment of bacteria to plant cell walls, thus initiating a recognition process that determines the host plant reaction (30). For example, the introduction of certain avirulent strains of the bacterial wilt pathogen, Pseudomonas solanacearum E. F. Smith, into tobacco leaves results in rapid attachment and envelopment of the bacteria by the host mesophyll cells (31). Virulent cells, on the other hand, remain free in the intercellular spaces and multiply rapidly during the first 36 to 48 h. The host component implicated in the initial attachment of bacteria to the plant cell wall is a glycoprotein that strongly agglutinates avirulent strains of P. solanacearum but does not agglutinate, or only weakly agglutinates, virulent strains (32). Originally, the bacterial agglutinin extracted from tobacco and potato tissues was thought to be the potato lectin described by Allen and Neuberger (3). Potato lectin has binding specificity for N-acetyl-D-glucosamine oligomers and is a strong hemagglutinin. When we attempted to purify the bacterial agglutinin from potatoes, however, most of the hemagglutinating activity was lost. Thus, it became apparent that the bacterial agglutinin and the lectin from potato tubers are different molecules. (23), as described by Sequeira and Graham (32). All procedures were done at 4°C. Peeled potato tubers were grated directly into two volumes of absolute ethanol: 1.25 N HCI (3:1). After 2 d, cellular debris was removed by filtering the mixture through cheesecloth and centrifuging the filtrate at 6,000g for 20 min. Then the supernatant was filtered through a Celite pad. Proteins were precipitated with three volumes of cold acetone. After 24 h, the precipitate was extracted with 0.1 M sodium acetate buffer (pH 3.6) at a ratio of 1 ml to 10 g of starting tissue. Undissolved material in the extract was removed by centrifugation (8,000g for 20 min) and the supernatant was dialyzed in se...

Chlamydomonas agglutinin is a hydroxyproline-rich glycoprotein

“…Noteworthy is the high content of hydroxyproline (=12%) and serine (--10%). These two amino acids serve as attachment points for oligosaccharide chains in cell wall glycopolypeptides of Chlamydomonas (13) and higher plants ( [14][15][16], in plant arabinogalactan proteins (17), and in the wall-associated lectins of the Solanacae (18)(19)(20)(21)(22)(23). Arabinans linked to hydroxyproline in plant cell wall proteins are thought to reinforce the polyproline II helical conformation (24, 25; G. J.…”

“…The self-assembly of Chlamydomonas wall glycoproteins into a crystalline pseudowall (42) undoubtedly involves molecular recognition, making it attractive to speculate that the sexual agglutinin has evolved from such structural proteins to perform an intercellular recognition or adhesion function. Because plant lectins also contain hydroxyproline-rich domains (18)(19)(20)(21)(22)(23), the Chlamydomonas agglutinin may well also prove to have lectin activity, with its stiff domain 2 serving to display more distal recognition domains to gametes of the opposite mating type. The presence of a collagen-like tail on the asymmetric form of-acetylcholinesterase of vertebrate nervous systems (43) and the crosslinking properties of the collagen IV component of the basal lamina (44) suggest that a similar molecular design may have been adopted more generally by proteins that function to mediate cell-cell interactions.…”

Metabolic changes in elicitor-treated bean cells. Enzymic responses associated with rapid changes in cell wall components

“…3), and this may be related to a later response characterised by increased levels of mRNA encoding the hydroxyproline-rich protein extensin (C. J. Lamb, personal communication). Further characterisation of the 42500-MI protein, which may prove to be an agglutinin similar to that from potato [6], is in progress. The bean 42 500-MI protein clearly differs fundamentally from extensin [45,461 and such a protein has not apparently been observed in other systems in which hydroxyproline proteins are induced in response to fungal infection; for example, preliminary data on de-glycosylated arabinosylated hydroxyproline proteins from melon suggest a peptide MI of 55000-60000 [47].…”

“…Induction of all these components therefore occurs as an early event in response to elicitor, although a later increase in proline 2-oxoglutarate dioxygenase activity occurs which is not accompanied by increases in the M , 42 500 protein; this later response may be related to accumulation of other hydroxyproline-rich components. 6 12 24…”

Serodiagnosis of pearl millet resistance to downy mildew by quantitating cell wall P/HRGP using polyclonal antiserum Pab-P/HRGP

“…Functionally they contribute to the mechanical properties of the cell wall by creating a network formed by glycoprotein elements and in addition they contribute to plant defence, by strengthening the wall upon pathogen attack or mechanical wounding. They may immobilize the pathogens by ionically binding to their negatively charged surfaces because of their positive charge (Leach et al 1982;Mazau et al 1987;Cassab and Varner 1988;Sommer-Knudsen et al 1998).…”