A hydroxyproline-rich protein in the soybean cell wall.

Abstract

A cDNA clone that hybridizes to an mRNA (lA10) that accumulates to a substantial level in the axis of the germinating soybean seed was sequenced. The amino acid sequence of the clone indicates an almost perfect repeat of Pro-Pro-Val-Tyr-Lys resulting in a protein containing 40% proline and lacking serine and histidine. On the likelihood that such a protein might be a hydroxyproline-rich cell-wall-glycoprotein (HRGP), cell walls of a soybean cell culture were extracted by procedures used to obtain soluble basic cell-wall glycoproteins, and the proteins were fractionated and purified. A 33-kDa protein (and possibly a 28-kDa protein) was obtained that has an amino acid distribution similar to that of the cDNA clone. The protein lacks histidine and serine and contains 20% hydroxyproline and 20% proline. The HRGP is thus distinct both in its amino acid content and in its pentameric repeat of Pro-Pro-Val-Tyr-Lys, with half of the prolines being hydroxylated. (4) was maintained on a 14-day cycle with a 1:12 dilution of cells (7 ml and 77 ml of medium in a 250-ml flask) every 2 weeks.In the preparation described in Results,25 ,uCi (1 Ci = 37 GBq) of [14C]proline was added to 8-day cells, and the cells were allowed to continue growing for 4 hr, after which time they were filtered on a coarse scintered funnel and frozen in liquid N2. In more recent preparations, 8-to 9-day cultures have been collected directly without labeling and the purification steps have been monitored solely by following the absorbance at 280 nm. Frozen cells (4.6 g) were homogenized in a Polytron in 0.25 M sucrose/3 mM EDTA/40 mM Tris-HCI, pH 8/0.5 mM phenylmethylsulfonyl fluoride/5 mM dithiothreitol (2.2 ml/g), and centrifuged for 2 min at 750 x g. The pellet was recentrifuged for 10 min at 23,500 x g and then washed one time in 14 ml of 5 mM potassium phosphate (pH 6.8) and six times with 15 ml of deionized water, centrifuging each time for 2 min at 750 x g. The pellet was extracted twice with 12 ml of 0.2 M CaC12 (5, 6), each extraction lasting 45 min. After centrifuging for 5 min at 2000 x g, the supernatant was adjusted with 68% trichloroacetic acid to a final concentration of 10% trichloroacetic acid, and the suspension was kept at 0C overnight. After centrifuging for 15 min at 24,000 x g, the supernatant was dialyzed against five changes of water over a 5-hr period and then overnight against 25 mM Tris HCI (pH 8.0). The dialyzer tubing had a Mr 3000 cutoff (Thomas 3787-H45). A typical preparation yielded 1.9 mg of protein based on 4 A280 units for a solution of 1 mg/ml, a figure calculated from the presence of 13 mol % tyrosine (see Table 1) in comparison with bovine serum albumin, which has 3.3% tyrosine (7) and 1 A280 unit for a solution of 1 mg/ml. The sample was chromatographed on a column of CM-cellulose (1.2 x 10 cm) equilibrated with 25 mM Tris-HCl (pH 8.0) and eluted with 100 ml of a linear gradient of 0.4 M NaCl in the same buffer. The 33-kDa protein, eluting at ""0.14 M NaCI, was pooled (tubes 17-27 in Fig. 3), concentra...

Characterization of two soybean repetitive proline-rich proteins and a cognate cDNA from germinated axes.

“…The amino acid content of the resolved RPRPs is given in Table 1. There are two significant differences from the data reported previously (Averyhart-Fullard, Datta, and Marcus, 1988): an increased level of glutamate and a somewhat reduced level of tyrosine. In addition, repeated analyses show a level of aspartate of 1 YO to 2.5%.…”

“…5-day to 12-day seedlings with a fragment containing the repeat region of the 1A10-2 cDNA (probe 1) or with the 1 A1 0-1 cDNA (Averyhart-Fullard, Datta, and Marcus, 1988) resulted in a high level of hybridization to a 1.1-kb to 1.2-kb mRNA ( Figure 5, probe 1). Because the IA1 0-1 cDNA (94% homologous to SbPRPl mRNA) is only 83% homologous to 1AlO-2, we initially interpreted the strong hybridization of this cDNA to indicate the presence of The sequences are aligned to give the maximal amino acid match.…”

“…Probe 1 is the 153 nucleotide AcclAccl fragment of 1A10-2 (Figure 3, 500 to 652). Identical data were obtained with the entire 1A10-2 cDNA or with the 1A10-1 cDNA (Averyhart-Fullard, Datta, and Marcus, 1988). Probe 2 is a synthetic oligomer corresponding to nucleotides 921 to 953 of SbPRPI.…”

“…The proteins have been isolated from wounded plant tissues (Stuart and Varner, 1980) and from cells in suspension culture (Smith, Muldoon, and Lamport, 1984) and contain Ser-(Hypk and Val-Tyr-Lys as the predominant repeats (Smith et al, 1986). Recently, we reported the isolation of two proteins (28 kD and 33 kD) from the walls of soybean cells in culture (Averyhart-Fullard, Datta, and Marcus, 1988). These proteins, representing a second class of hydroxyproline-rich cell wall protein, contain 20% proline and 20% hydroxyproline and appear to be related to cDNAs encoding sequences composed almost completely of repeats of Pro-Pro-Val-Tyr-Lys (SbPRPl , Hong, Nagao, and Key, 1987;1AlO-1, Averyhart-Fullard, Datta, and Marcus, 1988).…”

“…Figure 3 shows the nucleotide sequence of a clone (1A10-2) selected from this library. This clone, as well as two additional ones identical in sequence, were obtained using the I A 1 0-1 probe previously described (Averyhart-Fullard, Datta, and Marcus, 1988). The 1 A1 0-2 cDNA is clearly different in nucleotide sequence from the previously described cDNAs (IA1 0-1 and SbPRPl).…”