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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 19, 2004 as doi:10.1096/fj.03-0590fje. |
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Graduate School Neurosciences Amsterdam, Institute of Neuroscience, Departments of Molecular and Cellular Neurobiology; and
* Developmental Neurobiology, Faculty of Earth and Life Sciences, Vrije Universiteit, Amsterdam, The Netherlands
2Correspondence: Department of Molecular and Cellular Neurobiology, Vrije Universiteit, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands. E-mail: ronald.van.kesteren{at}falw.vu.nl
SPECIFIC AIMS
In the present study we aimed to identify novel internal defense peptides induced by parasitation. As a model we chose the intermediate snail host Lymnaea stagnalis of the schistosome parasite Trichobilharzia ocellata. Parasite-host interactions between these species have been well studied and the connective tissue of snail brains has proven to be a rich source of novel defense molecules. We tried to identify additional internal defense peptides.
PRINCIPAL FINDINGS
1. Granularin is up-regulated in cells of the snail internal defense system upon parasitation
We used mass spectrometric peptide profiling to identify peptides that are up-regulated during parasitosis in whole brain extracts, and then to assess their relative abundance in central ganglia (containing the neuronal cell bodies) and in peripheral nerves (containing large amounts of connective tissue). Our reasoning was that peptides that are equally abundant in ganglionic and nerve fractions are likely to be produced by the connective tissue cells rather than by neurons. One of the peptides that matched our criteria was further characterized and was named granularin after the granular cells of the nervous systems connective tissue that express it.
2. Granularin is a small peptide comprising a single vWF type C domain
Amino acid sequencing and cDNA cloning revealed that granularin is a secreted, 62 amino acid peptide that shares considerable sequence identity with the Von Willebrand factor (vWF) type C domain. In fact, Pfam database searching revealed that granularin contains a single vWF type C domain, flanked by only two amino acids at the N-terminus, and one amino acid at the C-terminus. The vWFC domain is an evolutionary conserved domain that is found in many secreted extracellular matrix proteins. Some have only a single copy of the vWFC domain (e.g., collagens and thrombospondins), others have multiple copies, such as neuralin (3 copies) and chordin (4 copies). The Kielin protein of Xenopus laevis even contains 22 vWFC domains. Some vWFC domain proteins have transmembrane domains, and may be involved in cell-to-cell communication and cellular adhesion (e.g., the cysteine-rich repeat protein crim-1 and the b0024.14 protein of C. elegans). Until now the vWFC domain has always been found in combination with other structural domains, giving rise to large proteins (e.g., mucins, >5,000 amino acids, the Von Willebrand factor, 2,813 amino acids, and the collagens, 
1,500 amino acids). Smallest of the vWFC domain proteins are the human connective tissue growth factor precursor and related proteins nov, cyr61 and wisp-1, which still carry additional structural domains and are approximately 360 amino acids in size. Thus, granularin represents a unique novel member of the vWFC domain protein family because of its small size and because of the sole appearance of a single vWFC domain.
3. Granularin gene expression is restricted to granular cells and up-regulated during parasitation
In situ hybridization on histological sections of the Lymnaea brain showed that cells in connective tissue surrounding the brain express the granularin gene. These cells were identified as the granular cells by phase contrast light microscopy based on the presence of large secretory granules. No expression could be detected in neurons. Reverse Northern blot was used to study the regulation of the granularin gene during parasitosis. Granularin mRNA levels showed a significant up-regulation of 100% as early as 5 h post-infection, which was sustained until 6 wks post-infection.
4. Granularin acts as an opsonin stimulating phagocytosis of foreign particles by hemocytes
Hemocytes are the main phagocytosing cells of the Lymnaea internal defense system. To study the effect of granularin on the phagocytosis, hemocytes were allowed to adhere to glass and zymosan particles were added. Under control conditions (i.e., without granularin), zymosan particles are readily phagocytosed by hemocytes, and the number of phagocytosing cells was easily counted (Fig. 1
A). Without granularin, 58% of hemocytes had phagocytosed zymosan within 30 min (Fig. 1B
). When hemocytes were first allowed to adhere to the glass, and zymosan and granularin were added together, no significant effect was observed on the number of phagocytosing cells (Fig. 1B
, bar 1). When adhered, hemocytes were preincubated with granularin before addition of zymosan, the number of phagocytosing cells was reduced to 38% (Fig. 1B
, bar 2), suggesting that blocking cell surface binding sites by granularin prevents phagocytosis. When we preincubated zymosan with granularin before addition to the hemocytes, the number of phagocytosing cells significantly increased to 69% (Fig. 1B
, bar 3). This effect must have been caused by granularin bound to zymosan particles since unbound granularin was removed from the solution. To further substantiate this conclusion, we incubated zymosan with FITC-labeled granularin. This resulted in brightly fluorescent particles (Fig. 1C
), whereas zymosan particles incubated with FITC-labeled insulin showed no labeling at all (Fig. 1D
). Together, these data are consistent with the idea that binding of granularin to foreign particles enables subsequent binding of these particles to hemocytes, which then induces phagocytosis, which is the classical definition of an opsonin raised by Wright and Douglas in 1903.
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CONCLUSIONS AND SIGNIFICANCE
We identified a novel molluscan opsonin that contains a single vWF type C domain, and of which the expression is up-regulated in granular cells during parasitosis. Granular cells contain large secretory granules that are released into the hemolymph by the process of exocytosis, and play an important role in the snail’s internal defense system. We previously identified the molluscan immunoglobulin MDM as one of the proteins secreted from the granular cells. Like granularin, MDM contains structural domains that are normally only found in large transmembrane proteins involved in cellular adhesion and cell-to-cell communication (i.e., five C2-like immunoglobulin domains). In another molluscan species, Biomphalaria glabrata, parasitation with the digenetic trematode Echinostoma paraensei results in the up-regulation of a family of lectin-like proteins named FREPs with an unusual domain organization. The use of protein domains derived from large complex proteins and recombined into novel peptides and proteins appears to be a common theme in the molluscan internal defense against endoparasites.
Because granularin is a secreted peptide containing a protein domain known to be involved in cellular adhesion, we wanted to test whether granularin might function as a soluble adhesion factor. In particular, we were interested in adhesive properties of granularin that might promote binding between hemocytes and foreign substances, and subsequent phagocytosis of these substances. Hemocytes are the most important circulating immune cells in the snail, and their phagocytotic activity is significantly enhanced during parasitosis. We used an assay in which hemocytes are immobilized on glass, and phagocytotic activity was measured by counting the number of cells that were able to take up zymosan particles (i.e., particles coated with bacterial antigens) in a certain amount of time. Our findings suggest that upon release, granularin first binds to foreign substances, enabling subsequent binding to hemocytes and phagocytosis. Thus, granularin acts as a classical opsonin (i.e., a serum factor that first binds to a foreign substance). This process, known as opsonization, renders the substance more susceptible to the phagocytosing capacity of blood cells. The exact mechanism by which this occurs in the case of granularin is not known. vWF itself plays an important role in blood clotting, and the type C domains in particular are involved in oligomerization of vWF protein by intermolecular disulphide formation. It has been suggested that other vWFC domain proteins are also involved in the formation of large protein complexes. Thus, it may well be possible that granularin oligomerizes around foreign particles, and that these larger oligomers are recognized by cell surface receptors on hemocytes and promote phagocytosis (Fig. 2
).
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In conclusion, we have identified a novel and unique member of the vWF type C domain protein family that is part of the internal defense response and specifically serves as an opsonin in phagocytosis of foreign particles by blood cells.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0590fje; 
This article has been cited by other articles:
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  P. M. Hermann, J. J. Nicol, A. G. M. Bulloch, and W. C. Wildering RGD-dependent mechanisms in the endoneurial phagocyte response and axonal regeneration in the nervous system of the snail Lymnaea stagnalis J. Exp. Biol., February 15, 2008; 211(4): 491 - 501. [Abstract] [Full Text] [PDF]
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