Fractionation of proteoglycans from bovine corneal stroma

Axelsson, Inge; Heinegård, Dick

doi:10.1042/bj1450491

Cited by 97 publications

(37 citation statements)

References 25 publications

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“…Proteins were isolated as described previously (41)(42)(43)(44). Human osteoadherin was expressed in human kidney cells, EBNA-293 (Invitrogen) by transfection with a pCEP4 vector (Invitrogen) with the transcription cytomegalovirus promoter included.…”

Section: Methodsmentioning

confidence: 99%

Identification of Tyrosine Sulfation in Extracellular Leucine-rich Repeat Proteins Using Mass Spectrometry

Önnerfjord

Heathfield

Heinegård

2004

Journal of Biological Chemistry

127

120

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Multiple and variable tyrosine sulfation in extracellular class II leucine-rich repeat proteins/proteoglycans were characterized by mass spectrometry. The sulfogroup on tyrosine is labile and is released from peptides under normal mass spectrometric conditions. Thus, special approaches must be considered in order to identify this modification. By using a combination of mass spectrometry studies operating in negative and positive ion mode, tyrosine sulfation could be identified. In positive mode, the peptides normally appeared non-sulfated, whereas in negative mode a mixture of sulfated and non-sulfated species was observed. A combination of peptides released by different proteinases was used to obtain details on the locations of sulfate groups. Multiple tyrosine sulfates were observed in the N-terminal region of fibromodulin (up to 9 sites), osteoadherin (up to 6 sites), and lumican (2 sites). Osteoadherin contains two additional sulfated tyrosine residues close to its C terminus. We also identified an error in the published sequence of bovine fibromodulin, resulting in the replacement of Thr 37 by Tyr 37 -Gly 38 , thus increasing its homology with its human counterpart.Tyrosine sulfation is a post-translational modification found on many secreted and membrane-bound proteins. As much as 1% of all tyrosine residues of the total protein in an organism can be sulfated, making this the most common post-translational modification of this residue (1). A number of proteins have been reported to contain sulfated tyrosines (2). Sulfation is suggested to occur at tyrosines located in close proximity to acidic residues (3). The existence of two different tyrosylprotein sulfotransferases, TPST-1 1 and TPST-2, might explain the diversity of sequences that are sulfated. Each enzyme may have a different substrate specificity and act on a different subset of target proteins. Tyrosine sulfation of chemokine receptor CCR-5 by TPST-1 and TPST-2 follows a discrete pattern and temporal sequence (4). The N terminus contains the sulfation sites with 0 -4 sulfogroups present. TPST-1 null mice (5) appeared healthy but had a ϳ5% lower average body weight than wild type animals. In addition, although the fertility of (Ϫ/Ϫ) males and females is normal, they have significantly smaller litters because of fetal death between 8.5 and 15.5 days postcoitum.Regulation of tyrosine sulfation in vivo by enzymatic sulfate removal is still largely unknown. However, human arylsulfatase A and B reside in the lysosome where they are thought to participate in the degradation of tyrosine sulfated proteins (6). In contrast, arylsulfatase E is found in the Golgi compartment (7). Mutations in this gene cause chondrodysplasia punctata, a congenital disorder characterized by abnormalities in cartilage and bone development (7-12).The biological role of tyrosine sulfation has been unclear, but recent studies have shown its functional importance in leukocyte adhesion (13-16), hormone synthesis (17-19), chemokine receptor signaling (20 -26), and hemostasis (27-33)....

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Section: Methodsmentioning

confidence: 99%

Identification of Tyrosine Sulfation in Extracellular Leucine-rich Repeat Proteins Using Mass Spectrometry

Önnerfjord

Heathfield

Heinegård

2004

Journal of Biological Chemistry

127

120

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“…Corneal proteoglycans are structurally very different from and smaller than cartilage proteoglycans (34,41,42) and have different protein compositions and far fewer glycosaminoglycan chains. These were not detected in situ by our antibodies to proteoglycan.…”

Section: Poole Et Almentioning

confidence: 99%

“…It has been shown to contain cartilage type II collagens (37)(38)(39)(40) in the sclera (but not in mammals), retina, and vitreous. The cornea (41)(42)(43) and sclera (44) contain proteoglycans that are structurally and functionally different from cartilage proteoglycans . In this study we demonstrate, using antibodies to native cartilage proteoglycan and link protein, that the sclera, but not the cornea, also contains molecules immunologically related to cartilage proteoglycans and link proteins and confirm that arterial vessels contain molecules immunologically related to cartilage link protein: these are present in the media.…”

mentioning

confidence: 99%

Mammalian eyes and associated tissues contain molecules that are immunologically related to cartilage proteoglycan and link protein.

Poole¹,

Pidoux²,

Reiner³

et al. 1982

The Journal of Cell Biology

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Monospecific antibodies to bovine nasal cartilage proteoglycan monomer and link protein were used to demonstrate that immunologically related molecules are present in the bovine eye and associated tissues. With immunofluorescence microscopy, reactions for both proteoglycan and link protein were observed in the sclera, the anterior uveal tract, and the endoneurium of the optic nerve of the central nervous system . Antibody to bovine nasal cartilage proteoglycan also reacted with some connective tissue sheaths of rectus muscle and the perineurium of the optic nerve of the central nervous system . .Antibody to proteoglycan purified from rat brain cross-reacted with bovine nasal cartilage proteoglycan, indicating structural similarities between these proteoglycans. ELISA studies and crossed immunoelectrophoresis demonstrated that purified dermatan sulphate proteoglycans isolated from bovine sclera did not react with these antibodies but that the antibody to cartilage proteoglycan reacted with other molecules extracted from sclera . Two molecular species resembling bovine nasal link protein in size and reactivity with antibody were also demonstrated in scleral extracts : the larger molecule was more common . Antibody to link protein reacted with the media of arterial vessels demonstrating the localization of arterial link protein described earlier. Tissues that were unstained for either molecule included the connective tissue stroma of the iris, retina, vitreous body, cornea, and the remainder of the uveal tract. These observations clearly demonstrate that tissues other than cartilage contain molecules that are immunologically related to cartilage-derived proteoglycans and link proteins .

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“…The extracellular matrix of the normal corneal stroma consists primarily of type I collagen and two proteoglycans: a chondroitin-dermatan sulfate proteoglycan and a keratan sulfate proteoglycan (4)(5)(6)(7)(8)(9). Because most of the sulfated carbohydrate in the cornea is contained in these two proteoglycans, macular corneal dystrophy may involve alterations in one or both of these proteoglycans.…”

mentioning

confidence: 99%

Macular corneal dystrophy: failure to synthesize a mature keratan sulfate proteoglycan.

Hassell¹,

Newsome²,

Krachmer³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

149

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Corneal specimens obtained during surgery from patients with macular corneal dystrophy and obtained at autopsy from control eyes were incubated in a medium containing radioactive precursors of glycoproteins and proteoglycans. Biosyntheticallyradiolabeled material was extracted and characterized by using molecular sieve chromatography and specific enzymes. Cells in control corneas synthesized both a chondroitin sulfate proteoglycan and a keratan sulfate proteoglycan similar to those present in monkey and bovine corneas. Cells in macular corneas synthesized a normal chondroitin sulfate proteoglycan but did not synthesize either keratan sulfate or a mature keratan sulfate proteoglycan. Instead, macular corneas synthesized a glycoprotein with unusually large oligosaccharide side chains. This glycoprotein was not detected in normal corneas and is slightly smaller than normal keratan sulfate prot can. The failure to synthesize a mature keratan sulfate proteoglycan may produce corneal opacity and result in blindness. Because of evidence indicating that the corneal keratan sulfate proteoglycan is normally synthesized through a glycoprotein intermediate [Hart, G. W. & Lennarz, W. (1978) J. Biol. Chem. 253,5795-5801, macular corneal dystrophy may be a defect in glycoprotein processing. Macular, lattice, and granular corneal dystrophies are characterized by specific abnormalities of the corneal stroma and result in loss of vision (1). Macular corneal dystrophy is characterized clinically by recessive inheritance, a general diffuse haze throughout the corneal stroma, and the accumulation of irregularly shaped deposits which tend to be anterior in the central region of the stroma. Histologically, the deposits are primarily extracellular and stain positively with Alcian blue and the periodic acid-Schiff reagent (2, 3). These observations suggest that the deposits may be glycosaminoglycans and oligosaccharides accumulating in the stromal extracellular matrix.The extracellular matrix of the normal corneal stroma consists primarily of type I collagen and two proteoglycans: a chondroitin-dermatan sulfate proteoglycan and a keratan sulfate proteoglycan (4-9). Because most of the sulfated carbohydrate in the cornea is contained in these two proteoglycans, macular corneal dystrophy may involve alterations in one or both of these proteoglycans. Recently, an organ culture system utilizing monkey corneas has been developed that allows the biosynthesis of radiolabeled corneal stromal proteoglycans in vItro (9). This system involves incubating intact corneas in a tissue culture medium containing radioactive precursors. Analyses of the radiolabeled proteoglycans synthesized during organ culture indicated that they were representative of the proteoglycans that accumulate in the stromal extracellular matrix.The corneal cloudiness in individuals with macular corneal MATERIALS AND METHODS Organ Culture Procedures. Corneas excised with a 7-to 8-mm trephine blade were obtained from human eyes at keratoplasty or from control donor eyes a...

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Fractionation of proteoglycans from bovine corneal stroma

Cited by 97 publications

References 25 publications

Identification of Tyrosine Sulfation in Extracellular Leucine-rich Repeat Proteins Using Mass Spectrometry

Identification of Tyrosine Sulfation in Extracellular Leucine-rich Repeat Proteins Using Mass Spectrometry

Mammalian eyes and associated tissues contain molecules that are immunologically related to cartilage proteoglycan and link protein.

Macular corneal dystrophy: failure to synthesize a mature keratan sulfate proteoglycan.

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