Detection and partial characterization of dissolved glycoproteins in oceanic waters

The widespread occurrence of dissolved proteins as well as porin proteins was confirmed in water columns at five stations located in the North Pacific, the northern North Pacific, and the Bering Sea, where no investigation has heretofore been made with regard to dissolved protein. The major dissolved proteins detected in this study were determined to be glycosylated, based on the detection of an aldehyde group formed by the periodate oxidation of the electrophoretically separated dissolved proteins. The cross reactivity against lectins to the glycoproteins indicated that there were two types of linkage between sugar chain and polypeptide; one was an N-linked sugar chain and the other was an O-linked sugar chain. In this study, a previously reported dissolved protein with an apparent molecular weight of 40 kilodaltons was identified as an OmpA-like protein, not a porin protein but a major outer membrane protein of most pathogenic gram-negative bacteria. The survival of porin proteins in dissolved organic matter is thought to give rise to the resistant structure of these proteins; consequently, unidentified dissolved proteins could also be porin proteins. However, the occurrences of glycoproteins and an OmpA-like protein demonstrated that proteins other than porin proteins account for the majority of dissolved proteins, since no glycosylated porin protein is known. There must therefore be an additional mechanism by which dissolved proteins are protected from biological attack. The possibility that glycoprotein sugar chains can help preserve other dissolved proteins in seawater, as well as the glycoproteins themselves, is discussed herein. Dissolved organic matter (DOM) in seawater plays an important role in marine ecosystems through the microbial loop (e.g., Azam 1998). Our knowledge of DOM has been rapidly increasing over the last decade (e.g., review by Ogawa and Tanoue [2003]); however, DOM is still the least understood organic reservoir in terms of its actual source, chemical nature, and formation processes. The ultimate source of DOM in the sea is the organic matter produced by phytoplankton (e.g., Nagata 2000). However, microbial derived D-enantiomers of amino acids were detected from DOM greater than 1 kilodalton (kDa) (McCarthy et al. 1998) and from bulk DOM (Dittmar et al. 2001). Detection of methylated and N-acetyl aminosugars from DOM greater than 1 kDa indicated a bacterial contribution to dissolved carbohydrate in seawater (Boon et al. 1998). Laboratory experiments also demonstrated that microbial activity played a role in the formation and determination of the chemical composition of DOM (Meon and Kirchman 2001; Ogawa et al. 2001). Growing evidence strongly indicates that bacterial organic constituents form a part of DOM. The occurrence of fewer than 30 dissolved proteins in oceanic seawater and the identification of porin proteins, which are trans–outer membrane channel proteins of gramnegative bacteria, provide direct evidence that bacterial mac1 Corresponding author ([email protected]). Acknowledgments We thank the onboard scientists and the captain and crew of RV Hakuho-maru for their cooperation during the KH95-3 and the KH99-3 cruises. We also thank S. Suzuki and T. Miyoshi for providing information on OmpA-like protein. We acknowledge two anonymous reviewers for their helpful comments. This work was partly supported by grants-in-aid for Scientific Research from the Ministry of Education, Science and Culture (13308029, 13878097, 14103001, 14209012), Japan. romolecules are a source of DOM greater than 10 kDa (Tanoue et al. 1995; Suzuki et al. 1997; Yamada et al. 2000). Many proteins in organisms are known to contain covalently bound sugars, termed glycoproteins, and their sugar chains have been shown to play a key function in complex cellular events (e.g., Winterburn and Phelps 1972). Glycoproteins are produced in marine environments; however, apart from a small number of porin homologue proteins, the chemical features, including glycosylation, of these dissolved proteins remain unknown. Plainly, further investigations are required to clarify the chemical nature and sources of dissolved pro-