Synthetic genes for glycoprotein design and the elucidation of hydroxyproline-O-glycosylation codes

Abstract

Design of hydroxyproline (Hyp)-rich glycoproteins (HRGPs) offers an approach for the structural and functional analysis of these wall components, which are broadly implicated in plant growth and development. HRGPs consist of multiple small repetitive "glycomodules" extensively O-glycosylated through the Hyp residues. The patterns of Hyp-O-glycosylation are putatively coded by the primary sequence as described by the Hyp contiguity hypothesis, which predicts contiguous Hyp residues to be attachment sites of small arabinooligosaccharides (1-5 Ara residues/Hyp); while clustered, noncontiguous Hyp residues are sites of arabinogalactan polysaccharide attachment. As a test, we designed two simple HRGPs as fusion proteins with green fluorescent protein. The first was a repetitive Ser-Hyp motif that encoded only clustered noncontiguous Hyp residues, predicted polysaccharide addition sites. The resulting glycoprotein had arabinogalactan polysaccharide O-linked to all Hyp residues. The second construct, based on the consensus sequence of a gum arabic HRGP, contained both arabinogalactan and arabinooligosaccharide addition sites and, as predicted, gave a product that contained both saccharide types. These results identify an O-glycosylation code of plants.

Figure 1

Oligonucleotide sets used to build the synthetic genes. Internal repeat oligonucleotide sets encoding Ser-Pro repeats or the gum arabic glycoprotein (GAGP) sequence were polymerized head-to-tail in the presence of the 5′ linker set. After ligation, the 3′ linker was added, and the genes were then restricted with BamHI and EcoRI and inserted into pBluescript II SK(+). The signal sequence was built by primer extension of the overlapping oligonucleotides featured here. The overlap is underlined.

Figure 2

Fluorescence micrographs of tobacco callus cells transformed with Sig-(Ser-Pro)₃₂-EGFP (A) or Sig-(GAGP)₃-EGFP (B); (C) nontransformed tobacco callus cells. The synthetic genes encoded a signal sequence to direct the products through the endoplasmic reticulum and Golgi, then out to the extracellular matrix (51). Not shown are cells transformed with Sig-EGFP, which looked like those in A and B; however, the medium fluorescence was much less intense. The fluorescence in these highly vacuolated, cultured cells surrounds the nuclei but is not inside of them, judging by optical sections (not shown). The microscope was a Molecular Dynamics Sarastro 2000 confocal laser scanning microscope with a 488-nm laser wave length filter, a 510-nm primary beam splitter, and a 510-nm barrier filter.

Figure 3

Superose-12 gel permeation chromatography with fluorescence detection of culture medium containing (Ser-Hyp)₃₂-EGFP (A), (GAGP)₃-EGFP medium concentrated 4-fold (B), medium of EGFP targeted to the extracellular matrix concentrated 10-fold (C), or 10 μg of standard EGFP from CLONTECH (D). Not shown is the fractionation of medium from nontransformed tobacco cells, which gave no fluorescent peaks, consistent with the results presented in Fig. 2C. LU, luminescence units.

Figure 4

PRP-1 reverse-phase fractionation of the Superose-12 peaks containing (Ser-Hyp)₃₂-EGFP (A), (GAGP)₃-EGFP (B), and (glyco)proteins in the medium of nontransformed tobacco cells (C). Endogenous tobacco AGPs eluted between 47 and 63 min; extensins eluted at ≈67 min. (C) Control medium collected from nontransformed tobacco cells was first fractionated on Superose-12, and the fractions eluting between 47 and 63 min were collected for further separation on PRP-1 to determine whether any endogenous AGPs/HRGPs cochromatographed with (Ser-Hyp)₃₂-EGFP or with (GAGP)₃-EGFP, which they did not.

Figure 5

Polypeptide sequences of (Ser-Hyp)₃₂-EGFP and (GAGP)₃-EGFP before and after deglycosylation. (A) N-terminal amino acid sequence of the glycoprotein (Ser-Hyp)₃₂-EGFP. We obtained partial sequence of both the glycoprotein (Upper) and its polypeptide after deglycosylation (Lower). X denotes blank cycles that correspond to glycosylated Hyp; glycoamino acids tend to produce blank cycles during Edman degradation, an exception being arabinosyl Hyp (17). (B) Polypeptide sequence of glycosylated (GAGP)₃-EGFP (Upper) and deglycosylated (GAGP)₃-EGFP (Lower). Residues marked with an asterisk denote low molar yields of Hyp and likely sites of arabinogalactan polysaccharide attachment in glycosylated (GAGP)₃-EGFP. For example, yields were 480 pM Asp in the first cycle, 331 pM Ser in the second, 194 pM Hyp in the third, and 508 pM Ser in the fourth.