Article |
Address correspondence to B.J. Hillier, University of California, San Diego, Scripps Institution of Oceanography, 9500 Gilman Dr., Mail code 0202, La Jolla, CA 92093. Tel.: (858) 534-2146. Fax: (858) 534-7313. E-mail: bhillier{at}ucsd.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: amino acid motifs; blood coagulation; disulfides; extracellular matrix proteins; immunology
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The literature from 1879 to 1959 on the clotting of coelomocytes of all echinoderm classes has been reviewed (Endean, 1966). Johnson (1969) summarized all previous work in this field and compared it to her observations on the behavior of sea urchin coelomocytes cultured in hanging drops. She concluded that clotting was a cellcell adhesion of the phagocytic coelomocytes, the other three types of cells were passively trapped in, or adhered to, the surfaces of clots. Other results showed that the cell-free plasma (CFP)* did not form a gel, and clot formation was irreversible. A calcium requirement for clotting had been demonstrated by others (Donnellon, 1938; Bookhout and Greenburg, 1940; Davidson, 1953). Clotting was also found to depend on the formation and presence of disulfide bonds (Boolootian and Giese, 1959; Bertheussen and Seijelid, 1978). More recent work showed that clotting was accompanied by a massive rearrangement of the actin cytoskeleton of the phagocytes that transformed from a petaloid to a filopodial morphology (Edds, 1977; Otto and Bryan, 1981).
The clotting of sea urchin coelomocytes is quite different from the mechanism of blood clotting in mammals (Doolittle, 1984), horseshoe crabs (Tai et al., 1977), and lobsters (Fuller and Doolittle, 1971) because unlike these other animals the protein concentration of sea urchin coelomic plasma is low (0.30.7 mg/ml) and varies among individuals and with the season (Holland et al., 1967). Also, >50% of the soluble coelomic protein is a molecule of 180 kD that is related to vertebrate transferrin (Brooks and Wessel, 2002) and has no clotting activity in our assay. We decided to study the biochemistry of clotting of sea urchin coelomocytes in an attempt to increase the general knowledge concerning different molecular mechanisms of intercellular adhesion.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Identification of the plasma factor
CFP from clot-inhibited WCF was obtained and subjected to ultracentrifugation (150,000 g for 1 h). Clotting activity was present in both the supernatant and the pellet; however, there appeared to be more activity in the pellet. SDS-PAGE separation of the proteins in the supernatant and the pellet showed that the pellet had a less complex protein composition. The ultracentrifuge pellets were solubilized in a buffered salt solution and subjected to anion exchange chromatography on DEAE-Sepharose. After washing and eluting with a linear salt gradient, an aliquot of each fraction was taken and assayed for clotting activity. Silver-stained gels of the eluted fractions (Fig. 3) showed that the coelomocyte clotting activity was associated with a diffuse band of Mr 75 kD (hereafter called amassin; fractions 1017). The purity of the 75-kD protein in some of the fractions (fractions 1216) suggested that it is the only plasma protein required for intercoelomocyte adhesion.
|
|
|
|
|
Cloning of the amassin cDNA
Using purified amassin, amino acid sequences were obtained from trypsin and cyanogen bromide-generated peptides. From this sequence, degenerate primers were designed (Table I) and used to obtain the complete 1,692 bp cDNA and derived amino acid sequences (sequence data available from GenBank/EMBL/DDBJ under accession no. AF533649). There is an 84-bp 5' untranslated region and a 123-bp 3' untranslated region containing a typical AATAAA polyadenylation signal 4 bp before the poly(A) tract begins. All seven sequenced peptides, totaling 112 aa, were found in the derived amino acid sequence of 495 residues, proving that the correct cDNA had been cloned. The antibodies made to native amassin specifically recognize a portion of the amassin sequence expressed recombinantly in bacteria (Fig. 8 A). Amassin has five potential N-linked glycosylation sites, which probably carry oligosaccharide chains, since digestion with the enzyme peptide N-glycosidase-F (PNGase-F) decreases amassin's Mr from 75 to 60 kD (Fig. 8 B), which is close to the calculated molecular weight of 56.5 kD. The remaining molecular weight discrepancy could be due to other posttranslational modifications such as O-linked glycosylation.
|
|
|
Cell surface receptor for amassin
Washed coelomocytes were treated with 0.1 mg/ml pancreatic trypsin for 30 min followed by the addition of soybean trypsin inhibitor to a final concentration of 0.2 mg/ml. Cell-free plasma (9 µg/ml) and calcium (10 mM) was then added, and the tubes were gently mixed for 10 min. The cells exposed to trypsin did not clot, suggesting that amassin binds to a cell surface protein (unpublished data). A control sample in which trypsin and the inhibitor were added together for 30 min clotted normally.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The fact that calcium ions are required for clotting was known previously (Donnellon, 1938; Bookhout and Greenburg, 1940; Davidson, 1953), and chelation of calcium has been employed to block clotting by those studying these cells (Edds, 1977). This requirement was also found in our work and used as a means to prevent clotting from occurring as a first step in studying the process of clotting. Whether the calcium requirement is involved in exocytosis of a needed molecule or plays a role in the maintenance of protein structure or enzyme activation remains unknown.
We have shown here that plasma-free coelomocytes, calcium, and amassin are the only three components needed for clotting in our in vitro assay. We cannot rule out the involvement of other minor proteins in the plasma, which might have been present in our purified amassin, although by silver-staining amassin appears highly purified (Fig. 3).
The mechanism that triggers clotting remains unknown. An uninjured animal must have a way to keep clotting from occurring. The washed coelomocytes might have a mechanism to become adhesion competent within seconds after injury to the coelomic cavity. Since treatment of coelomocytes with protease prevents their ability to clot, a cell surface protein seems to be involved. This could be a receptor protein which is exposed or activated on the surfaces of coelomocytes in response to injury. Integrins, which are expressed on coelomocytes (Burke, 1999), or cell surface lectins would be excellent candidate amassin receptors which could also regulate downstream signaling events.
Essentially nothing is known about the production of coelomocytes by the adult sea urchin. Although the antibody to amassin does not stain coelomocytes brightly and Western blots of coelomocytes show a weak reaction, amassin cDNA can be obtained from total coelomocyte RNA, indicating that at least some amassin could be synthesized in coelomocytes. Amassin appears to be a minor component of CFP, since Coomassie-stained SDS gels of CFP show only minor bands in the 75-kD region. The 180-kD transferrin-like protein (Brooks and Wessel, 2002) is >50% of the total protein in CFP; it does not possess clotting activity in our assay.
The OLF domain present in amassin is found in many extracellular proteins including the following: noelin, which has a neurotropic effect in development with a role in neural crest formation (Barembaum et al., 2000) and neurogenesis (Moreno and Bronner-Fraser, 2001); myocilin/TIGR, which is thought to have a role in the aqueous outflow of the vertebrate eye and is often mutated in some forms of glaucoma (Stone et al., 1997; Nguyen et al., 1998; Fautsch et al., 2000; Tamm and Russell, 2001); tiarin, a protein mediating dorsalization of the amphibian neural tube (Tsuda et al., 2002); and olfactomedin, a protein found in the mucus of bullfrog olfactory neuroepithelium (Snyder et al., 1991; Yokoe and Anholt, 1993). Although their roles are being elucidated, a function for proteins with an OLF domain has remained unknown.
The previous finding that coelomocytes fail to clot in the presence of n-ethyl maleimide (Bertheussen and Seijelid, 1978) and reducing agents (Boolootian and Giese, 1959) suggested that disulfide bond formation is required for clotting. These observations can be explained by the sensitivity of amassin to reductants. Clotting, allowed to proceed to completion, can be reversed by incubation with DTT and in the process release amassin as the dominant protein (Fig. 7 A). Amassin appears to exist as homooligomers held together by disulfide bonds; monomers, dimers, and higher multimers resolve on SDS-PAGE depending on the concentration of disulfide-reducing agent (Fig. 6). The coiled-coil regions of amassin could be involved in dimer formation. In myocilin/TIGR, the coiled-coil region has been shown by yeast two-hybrid analysis to possess dimerizing ability (Fautsch and Johnson, 2001; Wentz-Hunter et al., 2002). The oligomerization of the OLF family proteins TIGR (Nguyen et al., 1998) and olfactomedin (Snyder et al., 1991) has also been observed. Our structural analysis suggests that the other OLF family members (Fig. 9 C), known to reside in the extracellular matrix, might also be involved in formation of large polymer networks governing cell adhesion in a manner similar to amassin.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparation of washed coelomocytes and CFP
A three-step centrifugation method was used which sedimented enriched subpopulations of the four types of coelomocytes and were later combined. This was necessary to minimize damage to the fragile cells from excessive centrifugation, since the four types differ greatly in density. Clot-inhibited WCF was centrifuged (4°C) for 2 min at 150 g, which sedimented the red and colorless amoebocytes (also called spherule cells). The supernatant was then centrifuged at 150 g for 5 min to sediment the vibratile cells. Finally, the resulting supernatant was centrifuged at 600 g for 10 min to sediment the remainder of cells, mostly phagocytes (to prepare CFP, the supernatant above the sedimented phagocytes was centrifuged at 10,000 g for 20 min). After removal of the coelomic plasma from each enriched subpopulation, the cells were washed by resuspension in buffered calcium-free artificial seawater (10 mM Hepes, pH 7.5, 450 mM NaCl, 10.5 mM KCl, 26 mM MgCl2, 30 mM MgSO4, 2 mM NaHCO3) and resedimented as before. All three enriched subpopulations were resuspended in buffered calcium-free artificial seawater and combined at a concentration of 2 x 106 cells/ml.
Assay for clotting activity
Washed coelomocytes (1 ml) were combined with 20 µl of sample to be tested in a 1.5-ml microcentrifuge tube. One molar CaCl2 was then added to a final concentration of 10 mM followed by end-over-end rotation for 10 min. Qualitative scoring could be accomplished at this stage by visual inspection and comparison to controls (buffer only for the negative control and 10 µg unfractionated CFP for the positive control). For a quantitative measure of degree of clotting, after the 10-min rotation the partially clotted cell suspension was passed through a nylon mesh of 70 µm (Spectra). The light-scattering ability of the filtrate was then measured at 350 nm and normalized to the same controls to determine fraction clot completion.
Heat and trypsin treatment of CFP
Samples of CFP (0.5 mg/ml) were incubated for 10 min at 23, 37, 42, 50, 70, and 100°C and then added at a final concentration of 5 µg/ml to 1 ml washed coelomocytes and scored for activity. CFP was also incubated for 0, 15, 30, 60, 120, and 240 min in 0.1 mg/ml pancreatic trypsin at 23°C. Soybean trypsin inhibitor was added (0.2 mg/ml) to stop the digestion, and the clotting assay was performed.
Purification of amassin
The protein from the CFP of 10 sea urchins (400 ml) was precipitated by addition of ammonium sulfate to 70% saturation. After centrifugation at 10,000 g (20 min), the pellet was redissolved in 8 ml CIM and centrifuged at 150,000 g for 45 min. The resulting pellet was redissolved in 10 mM Tris, pH 8, 100 mM NaCl, 0.5 mM EDTA, and loaded onto 10 ml DEAE-Sepharose (Amersham Biosciences) equilibrated in the same buffer. After washing with 10 column vol, 3-ml fractions were collected during elution with a linear gradient of NaCl (1001,000 mM). Pure fractions of amassin were combined and concentrated by stirred cell ultrafiltration (Amicon) with a final yield of 0.4 mg. Estimating losses at all steps leads to an amassin protein concentration in the coelomic plasma at <1% of the total plasma protein. The concentration of total protein in CFP varies from 0.3 to 0.7 mg/ml.
DTT treatment to dissociate clots
Clots were dissociated into single cells by first washing them with excess filtered seawater and then incubating the washed clots in 0.45 µm filtered seawater containing 20 mM DTT and 20 mM Hepes, pH 8, with mild agitation for 20 min. Microscopic observation showed the coelomocytes were intact after dissociation. The cells were removed by sedimentation at 1,000 g for 10 min. The supernatant was then centrifuged at 10,000 g for 30 min to sediment debris. As a control, washed, clot-inhibited coelomocytes were treated in the same manner with DTT, and the supernatant was isolated.
PNGase-F digestion
Purified amassin (3 µg in 10 mM Tris, 200 mM NaCl, and 0.5 mM EDTA) was brought to 0.5% SDS and 50 mM ß-mercaptoethanol in a volume of 10 µl and then boiled for 5 min. Five µl of 7.5% NP-40 was added followed by 2 µl of PNGase-F and 13 µl water. The digestion was allowed to proceed overnight at 37°C.
Antibody preparation
Purified amassin (400 µg) was used to raise rabbit antiserum (Strategic Biosolutions). Antiserum was affinity purified on a column of bacterially expressed full-length amassin coupled to Affigel-10 following the manufacturer's directions (Bio-Rad Laboratories).
Cloning amassin sequence
Purified amassin was separated and excised from an SDS-PAGE gel and sent to the PAN Facility at Stanford University, where tryptic fragments were generated and sequenced. Additional CNBr-generated fragments were transferred to PVDF membrane and sequenced at the University of California at San Diego Protein Microsequencing Laboratory. Total RNA was isolated from an entire 7 g sea urchin by homogenization in a Waring blender with 40 ml of Trizol (Invitrogen) followed by centrifugation at 150 g for 5 min to clear debris. The supernatant was then processed following the manufacturers instructions. PolyA+ RNA was purified (Ambion), and first strand cDNA was synthesized with SuperScript II (Invitrogen) using random hexamers. Degenerate primers were designed from the peptide sequence and used to PCR amplify fragments of the amassin sequence (Table I). From the sequence of those PCR products, exact match primers were synthesized and used to complete the 1,692-bp amassin cDNA sequence by 5' and 3' rapid amplification of cDNA ends (Ambion). Sequence data for amassin is available from GenBank/EMBL/DDBJ under accession no. AF533649.
Western immunoblots
Proteins were resolved by SDS-PAGE and then transferred to Immobilon-P membrane (Millipore) in a tank transfer apparatus (Bio-Rad Laboratories). The membrane was blocked in 5% dry milk in TBST (50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween 20) for 1 h, followed by incubation with a 1:25,000 dilution (0.5 mg IgG/ml stock) of purified antiamassin in blocking buffer for 1 h, and washed four times for 15 min each. The membrane was then incubated with goat antirabbit IgG HRP-conjugated secondary antibody (Jackson ImmunoResearch Laboratories) at a 1:50,000 dilution in blocking buffer for 1 h, and washed four times for 15 min each. Amassin was detected with SuperSignal substrate (Pierce Chemical Co.).
Immunofluorescence
Coelomocytes in CIM were allowed to settle onto protamine-coated coverslips and were fixed for 45 min in seawater containing 20 mM Hepes, pH 7.5, 0.1% vol/vol glutaraldehyde and 3% wt/vol PFA. After washing, coverslips were blocked in TBST with 1% BSA and 5% normal goat serum. Affinity-purified antiamassin was used at various dilutions for 1 h. After washing, the secondary antibody, Alexa Fluor 546 goat antirabbit (Molecular Probes), was applied for 1 h at a 1:400 dilution. Negative controls of normal rabbit serum and no primary serum had absolutely no fluorescence after incubation with the Alexa Fluor secondary antibody.
Structural analysis
The five proteins (sequence data available from GenBank/EMBL/DDBJ) compared are: amassin from Strongylocentrotus purpuratus AF533649, noelin-1 from Gallus gallus Q9IAK4 (Barembaum et al., 2000), myocilin/TIGR from Bos taurus Q9XTA3 (Taniguchi et al., 2000), tiarin from Xenopus laevis BAB85495 (Tsuda et al., 2002), and olfactomedin from Rana catesbeiana Q07081 (Yokoe and Anholt, 1993). Assignment OLF domain positions were obtained with a Pfam search (Bateman et al., 2002). Secondary structure predictions were performed by using a sequence alignment of the OLF domains or the NH2-terminal ß-regions from the five proteins as input for the consensus method used by Jpred (Cuff et al., 1998). Signal peptides were predicted with the web server SignalP (Nielsen et al., 1997). The program Coils (Lupas et al., 1991) showed a high probability (>5 with a window of 28 residues) for the locations of the -helical coiled-coils.
![]() |
Footnotes |
---|
![]() |
Acknowledgments |
---|
This work was supported by National Institutes of Health grant HD12986.
Submitted: 10 October 2002
Revised: 2 January 2003
Accepted: 2 January 2003
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barembaum, M., T.A. Moreno, C. LaBonne, J. Sechrist, and M. Bronner-Fraser. 2000. Noelin-1 is a secreted glycoprotein involved in generation of the neural crest. Nat. Cell Biol. 2:219225.[CrossRef][Medline]
Bateman, A., E. Birney, L. Cerruti, R. Durbin, L. Etwiller, S.R. Eddy, S. Griffiths-Jones, K.L. Howe, M. Marshall, and E.L. Sonnhammer. 2002. The Pfam protein families database. Nucleic Acids Res. 30:276280.
Bertheussen, K., and R. Seijelid. 1978. Echinoid phagocytes in vitro. Exp. Cell Res. 111:401412.[Medline]
Bookhout, C.G., and N.D. Greenburg. 1940. Cell types and clotting reactions in the echinoid, Mellita quinquiesperforata. Biol. Bull. 79:309320.
Boolootian, R.A., and A.C. Giese. 1959. Clotting of echinoderm coelomic fluid. J. Exp. Zool. 140:207229.[Medline]
Brooks, J.M., and G.M. Wessel. 2002. The major yolk protein in sea urchins is a transferrin-like, iron binding protein. Dev. Biol. 245:112.[CrossRef][Medline]
Burke, R.D. 1999. Invertebrate integrins: structure, function, and evolution. Int. Rev. Cytol. 191:257284.[Medline]
Clow, L.A., P.S. Gross, C.S. Shih, and L.C. Smith. 2000. Expression of SpC3, the sea urchin complement component, in response to lipopolysaccharide. Immunogenetics. 51:10211033.[CrossRef][Medline]
Coffaro, K.A., and R.T. Hinegardner. 1977. Immune response in the sea urchin Lytechinus pictus. Science. 197:13891390.[Medline]
Cuff, J.A., M.E. Clamp, A.S. Siddiqui, M. Finlay, and G.J. Barton. 1998. JPred: a consensus secondary structure prediction server. Bioinformatics. 14:892893.[Abstract]
Davidson, E. 1953. Clotting of the perivisceral fluid of the sand dollar, Echinarachnius parma. Biol. Bull. 105:372.
Donnellon, J.A. 1938. An experimental study of clot formation in the perivisceral fluid of Arbacia. Physiol. Zool. 11:389398.
Doolittle, R.F. 1984. Fibrinogen and fibrin. Annu. Rev. Biochem. 53:195229.[CrossRef][Medline]
Edds, K.T. 1977. Dynamic aspects of filopodial formation by reorganization of microfilaments. J. Cell Biol. 73:479491.[Abstract]
Endean, R. 1966. The coelomocytes and coelomic fluids. Physiology of Echinodermata. R.A. Boolootian, editor. Interscience Publisher, New York. 301328.
Fautsch, M.P., and D.H. Johnson. 2001. Characterization of myocilin-myocilin interactions. Invest. Ophthalmol. Vis. Sci. 42:23242331.
Fautsch, M.P., C.K. Bahler, D.J. Jewison, and D.H. Johnson. 2000. Recombinant TIGR/MYOC increases outflow resistance in the human anterior segment. Invest. Ophthalmol. Vis. Sci. 41:41634168.
Fuller, G.M., and R.F. Doolittle. 1971. Studies of invertebrate fibrinogen. II. Transformation of lobster fibrinogen into fibrin. Biochemistry. 10:13111315.[Medline]
Gross, P.S., L.A. Clow, and L.C. Smith. 2000. SpC3, the complement homologue from the purple sea urchin, Strongylocentrotus purpuratus, is expressed in two subpopulations of the phagocytic coelomocytes. Immunogenetics. 51:10341044.[CrossRef][Medline]
Holland, L.Z., A.C. Giese, and J.H. Phillips. 1967. Studies on the perivisceral coelomic fluid protein concentration during seasonal and nutritional changes in the purple sea urchin. Comp. Biochem. Physiol. 21:361371.[Medline]
Johnson, P.T. 1969. The coelomic elements of sea urchins (Strongylocentrotus). I. The normal coelomocytes; their morphology and dynamics in hanging drops. J. Invertebr. Pathol. 13:2541.[Medline]
Lupas, A., M. Van Dyke, and J. Stock. 1991. Predicting coiled coils from protein sequences. Science. 252:11621164.[Medline]
Moreno, T.A., and M. Bronner-Fraser. 2001. The secreted glycoprotein noelin-1 promotes neurogenesis in Xenopus. Dev. Biol. 240:340360.[CrossRef][Medline]
Nguyen, T.D., P. Chen, W.D. Huang, H. Chen, D. Johnson, and J.R. Polansky. 1998. Gene structure and properties of TIGR, an olfactomedin-related glycoprotein cloned from glucocorticoid-induced trabecular meshwork cells. J. Biol. Chem. 273:63416350.
Nielsen, H., J. Engelbrecht, S. Brunak, and G. von Heijne. 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10:16.[Abstract]
Otto, J.J., and J. Bryan. 1981. The incorporation of actin and fascin into the cytoskeleton of filopodial sea urchin coelomocytes. Cell Motil. 1:179192.
Pancer, Z. 2000. Dynamic expression of multiple scavenger receptor cysteine-rich genes in coelomocytes of the purple sea urchin. Proc. Natl. Acad. Sci. USA. 97:1315613161.
Pancer, Z., J.P. Rast, and E.H. Davidson. 1999. Origins of immunity: transcription factors and homologues of effector genes of the vertebrate immune system expressed in sea urchin coelomocytes. Immunogenetics. 49:773786.[CrossRef][Medline]
Smith, L.C., R.J. Britten, and E.H. Davidson. 1992. SpCoel1: a sea urchin profilin gene expressed specifically in coelomocytes in response to injury. Mol. Biol. Cell. 3:403414.[Abstract]
Smith, L.C., L. Chang, R.J. Britten, and E.H. Davidson. 1996. Sea urchin genes expressed in activated coelomocytes are identified by expressed sequence tags. Complement homologues and other putative immune response genes suggest immune system homology within the deuterostomes. J. Immunol. 156:593602.[Abstract]
Snyder, D.A., A.M. Rivers, H. Yokoe, B.P. Menco, and R.R. Anholt. 1991. Olfactomedin: purification, characterization, and localization of a novel olfactory glycoprotein. Biochemistry. 30:91439153.[Medline]
Stone, E.M., J.H. Fingert, W.L. Alward, T.D. Nguyen, J.R. Polansky, S.L. Sunden, D. Nishimura, A.F. Clark, A. Nystuen, B.E. Nichols, et al. 1997. Identification of a gene that causes primary open angle glaucoma. Science. 275:668670.
Tai, J.Y., R.C. Seid, Jr., R.D. Huhn, and T.Y. Liu. 1977. Studies on Limulus amoebocyte lysate II. Purification of the coagulogen and the mechanism of clotting. J. Biol. Chem. 252:47734776.[Abstract]
Tamm, E.R., and P. Russell. 2001. The role of myocilin/TIGR in glaucoma: results of the Glaucoma Research Foundation catalyst meeting in Berkeley, California, March 2000. J. Glaucoma. 10:329339.[Medline]
Taniguchi, F., Y. Suzuki, H. Kurihara, Y. Kurihara, H. Kasai, S. Shirato, and M. Araie. 2000. Molecular cloning of the bovine MYOC and induction of its expression in trabecular meshwork cells. Invest. Ophthalmol. Vis. Sci. 41:20702075.
Tsuda, H., N. Sasai, M. Matsuo-Takasaki, M. Sakuragi, Y. Murakami, and Y. Sasai. 2002. Dorsalization of the neural tube by Xenopus tiarin, a novel patterning factor secreted by the flanking nonneural head ectoderm. Neuron. 33:515528.[CrossRef][Medline]
Wentz-Hunter, K., J. Ueda, and B.Y. Yue. 2002. Protein interactions with myocilin. Invest. Ophthalmol. Vis. Sci. 43:176182.
Yokoe, H., and R.R. Anholt. 1993. Molecular cloning of olfactomedin, an extracellular matrix protein specific to olfactory neuroepithelium. Proc. Natl. Acad. Sci. USA. 90:46554659.[Abstract]