Human unhydroxylated homotrimeric triple-helical collagen I produced in transgenic plants was used as an experimental model to provide insights into the role of hydroxyproline in molecular folding and fibril formation. By using chemically cross-linked molecules, we show here that the absence of hydroxyproline residues does not prevent correct folding of the recombinant collagen although it markedly slows down the propagation rate compared with bovine fully hydroxylated homotrimeric collagen I. Relatively slow cis-trans-isomerization in the absence of hydroxyproline likely represents the rate-limiting factor in the propagation of the unhydroxylated collagen helix. Because of the lack of hydroxylation, recombinant collagen molecules showed increased flexibility as well as a reduced melting temperature compared with native homotrimers and heterotrimers, whereas the distribution of charged amino acids was unchanged. However, unlike with bovine collagen I, the recombinant collagen did not selfassemble into banded fibrils in physiological ionic strength buffer at 20°C. Striated fibrils were only obtained with low ionic strength buffer. We propose that, under physiological ionic strength conditions, the hydroxyl groups in the native molecule retain water more efficiently thus favoring correct fibril formation. The importance of hydroxyproline in collagen self-assembly suggested by others from the crystal structures of collagen model peptides is thus confirmed experimentally on the entire collagen molecule.

Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.

Potential contamination of animal-derived collagen with pathogens has led to the demand for safe recombinant sources of this complex molecule. In continuation of our previous work [Ruggiero et al. (2000) FEBS Lett. 469, 132^136], here we show that it is possible to produce recombinant hydroxylated homotrimeric collagen in tobacco plants that are co-transformed with a human type I collagen and a chimeric proline-4-hydroxylase (P4H). This is to our knowledge the first time that transient expression in tobacco was used to improve the quality of a recombinant protein produced in plants through coexpression with an animal cell-derived modifying enzyme. We demonstrated the functionality of the new chimeric P4H and thus improved the thermal stability of recombinant collagen I from plants to 37³C. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Collagen is a potent adhesive substrate for cells, an event essentially mediated by the integrins ␣ 1 ␤ 1 and ␣ 2 ␤ 1 . Collagen fibrils also bind to the integrin ␣ 2 ␤ 1 and the platelet receptor glycoprotein VI to activate and aggregate platelets. The distinct triple helical recognition motifs for these receptors, GXOGER and (GPO) n , respectively, all contain hydroxyproline. Using unhydroxylated collagen I produced in transgenic plants, we investigated the role of hydroxyproline in the receptorbinding properties of collagen. We show that ␣ 2 ␤ 1 but not ␣ 1 ␤ 1 mediates cell adhesion to unhydroxylated collagen. Soluble recombinant ␣ 1 ␤ 1 binding to unhydroxylated collagen is considerably reduced compared with bovine collagens, but binding can be restored by prolyl hydroxylation of recombinant collagen. We also show that platelets use ␣ 2 ␤ 1 to adhere to the unhydroxylated recombinant molecules, but the adhesion is weaker than on fully hydroxylated collagen, and the unhydroxylated collagen fibrils fail to aggregate platelets. Prolyl hydroxylation is thus required for binding of collagen to platelet glycoprotein VI and to cells by ␣ 1 ␤ 1 . These observations give new insights into the molecular basis of collagen-receptor interactions and offer new selective applications for the recombinant unhydroxylated collagen I.The collagens include the most abundant proteins in mammalian tissues providing a scaffold and framework for extracellular matrix assembly. In addition, they can modulate cell behavior through interactions with cellular receptors. All members of the collagen family are built up of three chains and contain at least one triple helix domain formed by repeating GXY sequences (where X is often proline (P) and Y is often hydroxyproline (O)). Such molecules associate to form complex structures, of which the fibril-forming collagens constitute the most abundant matrix component (1). The higher order organization of the collagens, collagen I being the best example, proved crucial to the triggering of specific cell responses; native triple helical structure is essential for cell and platelet adhesion, and the fibrillar structure is required for platelet activation and aggregation (2).The cell surface receptors ␣ 1 ␤ 1 and ␣ 2 ␤ 1 of the integrin family are principally collagen receptors, although they can interact with other extracellular matrix components (e.g. laminins). The specificity of the more recently discovered collagen binding integrins, ␣ 10 ␤ 1 and ␣ 11 ␤ 1 , remains to be determined. Different receptor families have also been identified as containing collagen receptors, such as the membrane-spanning proteoglycans, syndecans (3), and the recently described tyrosine kinase receptors DDR1 and DDR2 (4). In platelets, the recently described glycoprotein VI is a key signaling collagen receptor and is an immunoglobulinn superfamily receptor (5).Several important clues to the understanding of the interaction of ␣ 2 ␤ 1 integrin with collagen arose from the development of triple helical synthetic peptid...

Aims:The cell envelope of the fish pathogen Flavobacterium psychrophilum contains more than 50 polypeptides resolved by sodium dodecyl sulphate-polyacrylaminde gel electrophoresis analysis including a major component named P60. Here, we have developed a simple and efficient procedure for the purification of P60 and therefore permitting its biochemical characterization. Methods and Results: Membrane proteins were selectively extracted from isolated cell envelopes with the mild non-ionic detergent Triton X-100. About 10 polypeptides were identified from the detergent fraction, including P60. The P60-enriched fraction was thereafter subjected to an anion exchange chromatographic step in the presence of Triton X-100. The molecule was purified at the milligram level (yield, about 75%; purification factor, 6.2). Analyses performed by charge shift electrophoresis, Triton X-114 phase separation and by detection of sugarmodified components showed that P60 is a true amphiphilic membrane-associated glycoprotein. Conclusions: The method described in this paper provides pure and non-denaturated P60 and should prove to be easily scaled-up. As sugar-modified protein, P60 should be included in the growing list of glycosylated prokaryotic proteins. Significance and Impact of the Study: It offers the possibility of obtaining P60 in amounts allowing the testing of the potential of P60 as a candidate for anti-flavobacteria subunit vaccines, as P60 is one of the major antigens.