Characterisation of a developmentally related polypeptide with glutelin solubility characteristics from Lupinus albus L.

Proteins from Lupinus albus L. cv. Rio Maior seeds were fractionated according to solubility criteria. Patterns of concanavalin A (ConA)-binding polypeptides from the different classes, albumins, globulins, glutelins and prolamins, were established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two bands of apparent molecular masses of 29 and 23.5 kDa with glutelin solubility characteristics bound the lectin. The 23.5-kDa band was separated by two-dimensional electrophoresis into two components: one glycosylated and heterogeneous with an isoelectric point of approx. 10 (designated as G23) and another, not detected with ConA, precipitating in the first dimension. The amino acid and hexosamine analysis of G23 showed that it is particularly rich in Gly (11.2%), Glx (10.0%), Ser (9.0%), Leu (8.2%), Asx (7.5%), and Pro (6.7%) and that it has a considerable content of the sulphur-containing amino acids Met (2.0%) and Cys (5.8%) and contains glucosamine. The determined N-terminal amino acid sequence of G23 was: ¹KG(R)V⁵KGTGD¹⁰(T)PXXV¹⁵XLY(N)R²⁰T, and this had no significant similarity to any of the amino acid sequences contained in the data bank SWISS-PROT 26. The glycoprotein G23 was completely deglycosylated with peptide-N-glycosidase F, yielding a homogeneous 21-kDa polypeptide composed of approximately 191 amino acids. The structures of the major N-linked neutral oligosaccharides of G23, determined by exoglycosidase sequencing, were as follows: Man2Man6(Man3) Man6(Man2Man2Man3)Man4GlcNAc4GlcNAc (13%); ± Man2Man6(Man3)Man6(± Man2 Man2 Man3)Man4GlcNAc4GlcNAc (29%); Man6(Man3) Man6(Man2Man3)Man4GlcNAc4GlcNAc (13%); Man6(Man3)Man6(Man3)Man4GlcNAc4GlcNAc (16%); Man6(Man3)(Xyl2)Man4GlcNAc 4GlcNAc (28%). Changes in G23 abundance during seed development, germination and seedling growth were monitored with a specific antibody. The glycoprotein G23 started to accumulate appreciably during seed formation between the 40th and the 50th days after anthesis and was detected following seed imbibition, until the 9th day in cotyledons, the 2nd day in roots and the 4th day in hypocotyls and leaves.Abbreviations ConA concanavalin A - Endo H endo-N-acetyl--d-glucosaminidase H - GlcNAc N-acetylglucosamine - gu glucose unit - IEF isoelectric focusing - Man mannose - NEPHGE non-equilibrium pH gradient electrophoresis - PNGase F peptide-N-glycosidase F - PVDF polyvinylidenedifluoride - Xyl xyloseWe thank Geoffrey Guile (Oxford Glycobiology Institute, Oxford, UK) for help with HPLC separations and amino acid and hexosamine analysis, Terry Butters (Oxford Glycobiology Institute) for providing the exoglycosidases and advice in their use, Manuela Regala (Instituto de Tecnologia Química e Biológica Oeiras, Portugal) and Paula Veríssimo (University of Coimbra, Portugal) for determining the N-terminal amino acid sequence of G20 and G23 and Dr. Jorge Lampreia (Universidade Nova de Lisboa, Lisbon, Portugal) for the computerised search of the SWISS-PROT data bank. Lupinus albus seeds were provided by Dr. João Neves Martins (Instituto Superior de Agronomia Lisbon, Portugal). We also thank J. Romão (Instituto Gulbenkian de Ciência, Oeiras, Portugal) for technical assistance in antibody production. This work was supported by Junta National de Investigação Científica e Tecnológica, Portugal.