Oligosaccharide Sequence of Human Breast Cancer Cell Heparan Sulfate with High Affinity for Laminin*

Narayanan ParthasarathyDagger §, Lisa F. GotowDagger , James D. BottomsDagger , Timothy E. Kute, William D. WagnerDagger , and Barbara Mulloyparallel

From the  Department of Pathology and Dagger  Section of Comparative Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157-1040 and the parallel  National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Harts, En6 3QG, United Kingdom

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Laminin-1 is a basement membrane glycoprotein implicated in tumor-host adhesion, which involves the cell-binding domain(s) of laminin-1 and tumor cell surface heparan sulfate (HS). The specific tumor cell surface HS oligosaccharide sequences that are necessary for binding to laminin-1 have not been characterized. To identify this laminin-binding oligosaccharide sequence, GlcNSO4-rich oligosaccharides terminating with [3H]2,5-anhydromannitol (AManR) residues were isolated from human breast cancer cell (MCF-7)-derived HS through hydrazinolysis/high pH (4.0) nitrous acid treatment/[3H]NaBH4 reduction. These oligosaccharides were chromatographed on a laminin-1 affinity column. A high affinity dodecasaccharide was isolated and characterized. Disaccharide analysis yielded IdoA(2-SO4) right-arrow AManR(6-SO4) as the only disaccharide upon treatment of this dodecasaccharide with nitrous acid at low pH (1.5). The sequence of laminin-binding high affinity oligosaccharide is therefore [IdoA(2-SO4) right-arrow GlcNSO4(6-SO4)]5[IdoA(2-SO4) right-arrow AManR(6-SO4)]. Low affinity dodecasaccharides composed of [IdoA(2-SO4) right-arrow GlcNSO4(6-SO4)]5, [IdoA(2-SO4) right-arrow GlcNSO4] were also isolated by laminin-1 affinity chromatography. Molecular modeling studies indicate that a heparin-binding peptide sequence corresponding to amino acid residues 3010-3031 (KQNCLSSRASFRGCVRNLRLSR) in the G domain of laminin-1, modeled as a right-handed alpha -helix, carries an array of basic residues well placed to bind to clusters of sulfate groups on the high affinity dodecasaccharide.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Heparan sulfate (HS)1 is a glycosaminoglycan polymer consisting of sequences of uronic acid-glucosamine disaccharides (1) in which uronic acid may be either glucuronic or iduronic acid and the glucosamine residue may be either N-acetylated (GlcNAc) or N-sulfated (GlcNSO4). Structural studies (2) indicate that blocks of GlcNSO4 containing sulfate-rich disaccharides ("heparin-like" domains) are interspersed with blocks of sulfate-poor domains containing GlcNAc disaccharides. The latter domains might also be composed of unsubstituted glucosamine-containing domains (3). Thus, heparan sulfate is a multidomain polysaccharide. These domains, with different structural modifications including O-sulfation of uronic acid residues and N- and O-sulfation of glucosamine residues, impart specific properties to heparan sulfate. For example, 3-O-sulfated glucosamine and 2-O-sulfated hexuronic acid residues have been implicated in antithrombotic (1) and mitogenic activities of bFGF-induced cell proliferation (4). Heparin-like domains of HS are also implicated in binding to a number of biologically important molecules such as lipoprotein lipase (5, 6), hepatocyte growth factor (7), and platelet factor 4 (8). Heparin as well as HS is also known to bind to laminin-1 (9, 10), a basement membrane glycoprotein postulated to be involved in tumor-host adhesion in metastasis. It is likely that sulfate groups of heparin/HS might be involved in binding to a region(s) of basic amino acids in laminin-1 with high affinity. However, no information is available on the nature of the laminin-binding oligosaccharide sequences in tumor cell HS. In the present study, we report the identification of a distinct oligosaccharide structure in human breast cancer cell (MCF-7) HS that exhibits strong affinity for laminin-1.

    EXPERIMENTAL PROCEDURES
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Introduction
Procedures
Results
Discussion
References

Cell Culture-- Human breast cancer cells (MCF-7) were purchased from American Type Culture Collection (Rockville, MD). Cells were cultured in MEM with Earle's salts containing non-essential amino acids, 2 mM glutamine, 10% fetal bovine serum, 1 mM sodium pyruvate, bovine insulin (10 µg/ml), and penicillin (100 units/ml)/streptomycin (100 µg/ml) in 225-cm2 flasks, incubated at 37 °C with 5% CO2.

Preparation of Laminin-1 Affinity Column-- Mouse laminin-1 (nidogen-free) was purchased from ICN. Laminin-1 was coupled to Reacti-Gel (6×) Support according to manufacturer's instructions (Pierce), in the presence of heparin (Sigma, Grade I) (laminin:heparin ratio = 1:30, by weight). The efficiency of coupling was approximately 80%. The laminin affinity column prepared in this manner contained 0.8 mg of laminin/ml of gel.

Preparation of MCF-7 Cell-associated HS-- MCF-7 cells were biosynthetically radiolabeled (11) with 20 µCi/ml carrier-free [35S]Na2SO4 (~43 Ci/mg of sulfur) (ICN Pharmaceuticals Inc.) for 48 h. The medium was removed, and cells were washed twice with phosphate-buffered saline. The cell layer was solubilized with cold 0.1 M NaOH for 15-20 min. An aliquot of the cell fraction was assayed for protein content (12), and the remainder was adjusted to pH 8.0 by adding 50% (v/v) acetic acid. After adjusting to 5 mM CaCl2, the material was digested with 2% w/w Pronase (Calbiochem) at 37 °C for 48 h. The digest was diluted by 5 volumes with water and subjected to purification by DEAE-Sephacel (Amersham Pharmacia Biotech) filtration (13). The sample was applied to the column (5 ml bed volume), which was equilibrated with 20 mM sodium acetate buffer, pH 6.5, containing 0.25 M NaCl. After washing the column with 20 volumes of the equilibrating buffer, bound glycosaminoglycan was eluted with 10 column volumes of 2 M NaCl in 20 mM sodium acetate buffer, pH 6.5. The glycosaminoglycan material was recovered by extensive dialysis using Spectraphore Membrane (Mr cut off 3500) against water, followed by lyophilization. The purified glycosaminoglycan was digested with chondroitinase ABC lyase (Seikagaku America Inc.) (14). The digest was fractionated on a DEAE-cellulose column (3 ml bed volume) in pyridine-formate buffers (15). After removal of chondroitin sulfate-derived disaccharides by washing with 0.5 M pyridine acetate, pH 5.0, [35SO4]HS peptides were eluted with 2.5 M pyridine acetate, pH 5.0. [35SO4]HS peptides were subsequently subjected to beta -elimination (1 M NaBH4, 50 mM NaOH, 45 °C, 20 h) to release HS chains (16). Excess borohydride was destroyed by addition of ice-cold 2 M acetic acid, and HS chains were recovered after passage through AG-50W-X8 (200-400 mesh H+ form) (Bio-Rad). 35SO4 activity was monitored during HS isolation only, whereas 3H activity was monitored during the subsequent fractionation of 3H-oligosaccharides.

Preparation of MCF-7 Cell-associated HS Oligosaccharides by Hydrazinolysis-- Hydrazinolysis of HS chains was carried out by treatment with anhydrous hydrazine, 1% hydrazine sulfate for 9 h at 100 °C as described previously (5, 17). The deacetylated HS chains were treated with high pH (4.0) nitrous acid reagent (0.5 M H2SO4, 5.5 M NaNO2, 2:5) for 30 min at room temperature (18). The pH of the oligosaccharide products was adjusted to 8.5 with 1 M Na2CO3 and reduced with 0.5 mCi of [3H]NaBH4 (1.5 Ci/mmol) (ICN) at 50 °C for 30 min (5). The labeled oligosaccharides were recovered by passage through AG-50W-X8 (200-400 mesh, H+form), as described previously.

Chromatography of MCF-7 HS Oligosaccharides on Laminin-1 Affinity Column-- 3H-Labeled HS oligosaccharides were loaded onto a 1-ml laminin-1 affinity column, which was equilibrated with 2 mM sodium acetate buffer, pH 6.5, 5 mM CaCl2, 5 mM MgCl2. The flow-through was reloaded onto the column three to four times to achieve maximal binding. The column was washed with 30 ml of equilibrating buffer containing 140 mM NaCl. Bound oligosaccharides were eluted in a stepwise manner with the buffer containing 0.4 and 1.5 M NaCl. Fractions of 1 ml were collected, and the radioactivity in an aliquot of each fraction was determined.

Gel Chromatography-- Gel chromatography of laminin-bound 3H-labeled HS oligosaccharides was performed on a Bio-Gel P-10 (Bio-Rad) column (1.5 × 85 cm) in 0.5 M pyridine acetate, pH 5.0 (13); 1-ml fractions were collected.

Disaccharide Analysis-- The laminin-bound oligosaccharides were converted to disaccharides by low pH (1.5) HNO2 treatment (19) and reduced with [3H]NaBH4. The disaccharides were further purified by passage through Darco-Celite columns (20) and then analyzed on a 4.6 × 250 mm Partisil 10 SAX column (Whatman) using a stepwise gradient of 12 mM, 26 mM, and 200 mM KH2PO4 to elute nonsulfated, monosulfated, and disulfated disaccharides, respectively. The sulfated 3H-disaccharide mixture standard, from heparin, was purchased from Chirazyme Inc., Urbana, IL.

Molecular Modeling-- Molecular models were built (5, 21) and visualized using the program Insight (MSI, San Diego, CA).

    RESULTS
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Abstract
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Procedures
Results
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References

Laminin Affinity Fractionation of MCF-7 Cell-associated HS Oligosaccharides-- Isolated MCF-7 HS chains were N-deacetylated by hydrazinolysis and reacted with nitrous acid at high pH (4.0) (18) to cleave the glycosidic linkages following N-unsubstituted D-glucosamine residues. The resulting oligosaccharide mixture, terminating at 2,5-anhydromannose residues, was reduced with [3H]NaBH4 to produce 3H-oligosaccharides, which were fractionated on a laminin-1 affinity column. Approximately 97% of the oligosaccharides did not bind to the column. Bound oligosaccharides, constituting 3% of the total radioactivity loaded, were eluted from the column in the 0.4 and 1.5 M NaCl fractions (Fig. 1).


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Fig. 1.   Affinity chromatography of 3H-labeled MCF-7 cell HS-derived oligosaccharides on a laminin-1 affinity column. 3H-Labeled HS-derived oligosaccharides were applied to a laminin-1 affinity column as described under "Experimental Procedures." Bound material (approximately 3% of total dpm applied) was eluted with a stepwise gradient of NaCl.

The size of 3H-labeled HS oligosaccharides with high (1.5 M NaCl) and low (0.4 M NaCl) affinity for laminin-1 was determined by Bio-Gel P-10 chromatography. High as well as low affinity oligosaccharides were predominantly comprised of dodecasaccharides (6 disaccharides in length), as they were eluted in a position between low molecular weight heparins of Mr ~5000 and Mr ~3500 (Fig. 2).


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Fig. 2.   Bio-Gel P-10 chromatography of HS oligosaccharides with low and high affinity for laminin-1. 3H-Labeled MCF-7 HS-derived low and high affinity oligosaccharides were examined for their size by gel filtration on a Bio-Gel P-10 column (1.5 × 85 cm) in 0.5 M pyridine acetate, pH 5.0. The void volume (1) and the position of elution of heparin (Mr = 5000) from Calbiochem (2) and heparin (Mr = 3500) from Sigma (3) are indicated by arrows.

Disaccharide Composition of Laminin-1-bound Oligosaccharides-- Low pH (1.5) nitrous acid treatment was employed to degrade high affinity oligosaccharides into disaccharides, which were further reduced with [3H]NaBH4. 3H-Disaccharides were subsequently analyzed by ion-exchange HPLC on a Partisil 10 SAX column eluted with a KH2PO4 gradient. By comparison with standard disaccharides from commercial heparin (Fig. 3A) and with published data (18), ISMS was found to be the only major disacccharide (Fig. 3B) of the laminin-1 high affinity oligosaccharide. Based on these findings, the probable structure for laminin-1 high affinity oligosaccharide is [IdoA(2-SO4) right-arrow GlcNSO4(6-SO4)]5[IdoA(2-SO4) right-arrow AManR(6-SO4)]. The low affinity dodecasaccharide gave rise to two disaccharides, ISMS and ISM at the ratio of 5:1 (data not shown).


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Fig. 3.   HPLC ion-exchange chromatography of disaccharides from nitrous acid-degraded laminin-1 high affinity dodecasaccharides from MCF-7 cell HS. Elution profile of disaccharides from high affinity MCF-7 HS dodecasaccharides (B) compared with standard 3H-labeled disaccharide mixture (A) from Chirazyme Inc. 1, GSM; 2, GMS; 3, IMS; 4, ISM; 5, ISMS. The low affinity dodecasaccharide fraction contained ISMS and ISM as their disaccharides in the ratio of approximately 5:1 (data not shown).

Molecular Modeling of Laminin-1 High Affinity Dodecasaccharide and Its Interaction with the G Domain of Laminin-1-- A molecular model of a heparin dodecasaccharide, corresponding with the high affinity structure described above, was obtained by using the atomic coordinates of an NMR-derived solution conformation of heparin (21). Heparin binding activity has been localized to the G domain of recombinant laminin-1 (22), and a peptide sequence in this region, amino acids 3010-3031 (KQNCLSSRASFRGCVRNLRLSR), has been suggested as a heparin-binding site (23). A three-dimensional structure has not been reported for the G domain of laminin-1, and the above sequence does not show sufficiently strong similarity to any peptide segment of known structure to allow modeling on the basis of homology. However, the sequence does contain several basic residues spread along its length, and when the sequence is modeled as a right-handed alpha -helix all four internal arginine residues are found on one side of the helix (Fig. 4A). This configuration of the peptide would allow the flexible arginine side chains to interact with clusters of sulfate groups along the length of the high affinity oligosaccharide (Fig. 4B).


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Fig. 4.   Molecular models of a suggested heparin-binding sequence in the G domain of laminin-1 and of a dodecasaccharide heparan sulfate sequence with high affinity for laminin-1. The heparin binding sequence in the G domain of laminin-1, KQNCLSSRASFRGCVRNLRLSR, modeled as a right-handed alpha -helix, has four internal arginine residues disposed in an approximately linear fashion down one side of the helix (A). The heparan sulfate dodecasaccharide with high affinity for laminin, modeled on the basis of the NMR structure of the same sequence in heparin (21), shows clusters of sulfate groups arranged down the sides of the heparan sulfate molecule (B). The basic residues in the alpha -helical peptide are well placed to interact with the sulfate clusters.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
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References

The present investigation shows that a sulfate-rich oligosaccharide fraction in MCF-7 tumor cell HS, composed of a sequence of six disaccharides, binds strongly to laminin-1. This oligosaccharide was generated by employing a hydrazinolysis/deamination procedure (18), which causes the cleavage of deacetylated N-acetylglucosaminic bonds at pH 4.0 and releases HS fragments rich in N-sulfated glucosamine residues. This high affinity oligosaccharide (Fig. 1) consists of six repeating disaccharide units of the structure [IdoA(2-SO4) right-arrow GlcNSO4(6-SO4)] (Figs. 2 and 3B). Therefore, the structure for laminin-bound high affinity oligosaccharide is [IdoA(2-SO4) right-arrow GlcNSO4(6-SO4)]5[IdoA(2-SO4) right-arrow AManR(6-SO4)]. A slightly less sulfated dodecasaccharide of the composition [IdoA(2-SO4) right-arrow GlcNSO4(6-SO4)]5[IdoA(2-SO4) right-arrow GlcNSO4] was also isolated. While the homogeneity of this oligosaccharide remains to be ascertained, it binds to laminin-1 with low affinity (Fig. 1) compared with the high affinity oligosaccharide, even though both of these oligosaccharides are approximately the same size (Fig. 2).

The strong interaction of laminin-1 with heparin or HS has been known (9) for quite some time. Indeed, heparin-agarose chromatography has been routinely employed (24) to separate laminin-1 subunits. Despite reports suggesting a strong binding between laminin-1 and heparin or heparan sulfate, to our knowledge, this is the first report of a specific extended oligosaccharide sequence in HS that has high affinity for laminin-1.

A number of heparin-binding proteins, comprised of different biologically important macromolecules, including bFGF (25), hepatocyte growth factor (26), platelet factor 4 (8), and lipoprotein lipase (5) interact with heparan sulfate via specific oligosaccharide sequences enriched in IdoA(2-SO4) right-arrow GlcNSO4(6-SO4). Along with N-sulfation of glucosamine residues, 2-O-sulfation of iduronic acids and/or 6-O-sulfation of glucosamine residues are the necessary structural requirements for these heparin-binding proteins to interact and elicit their biological activities. In the present study, a similar oligosaccharide sequence was also involved in the binding of HS to laminin-1. Experiments are in progress to examine the minimal sulfation requirements of HS for binding to laminin-1, through chemical modification of laminin affinity oligosaccharide. Alternatively, Chinese hamster ovary cell mutants defective in heparan sulfate biosynthesis (13, 27) could be employed to study the role of sulfation in interaction and adhesion to laminin-1.

Heparan sulfate is also known to bind to a number of biologically important molecules (28) including antithrombin (1), bFGF (25), hepatocyte growth factor (26), and lipoprotein lipase (5). The binding sites, or the percent of oligosaccharides with affinity for these proteins, constitute less than 5% of the total oligosaccharides (1, 5). Thus, in the present study it is not surprising that laminin-1 bound to highly specific oligosaccharide sequences, which comprise only 3% of the total MCF-7 oligosaccharides. It is possible that even though present in low proportions, the strategic locations of these binding regions and concentration on the tumor cell surface would favor their interaction with laminin-1. Highly sulfated, iduronate-rich heparan sulfate oligosaccharide sequences such as these can be expected to adopt the characteristic heparin conformation (21), in which clusters of sulfate groups are formed and are favorable to bind to basic amino acids on the protein surface. Modeled as an alpha -helix, the heparin-binding sequence in the G domain of laminin-1 (Fig. 4A) offers a linear array of basic residues well-placed to interact with these sulfate clusters (Fig. 4B). Amphipathic alpha -helical peptides with high heparin affinity have previously been described in other proteins (29). This model of the heparin/laminin interaction remains tentative, and it is insufficiently detailed to explain the difference in affinity between the fully sulfated and slightly undersulfated dodecasaccharide fragments. The full heparin-binding site of laminin may also be more extensive, involving discontinuous segments of peptide sequence (30).

A family of cell surface transmembrane HS proteoglycans, called syndecans (syndecan I through IV), have been reported to be synthesized by a variety of cells (31, 32). In syndecans, HS chains are generally composed of a series of conserved sulfate-rich (mainly N-sulfated) domains, containing IdoA(2-SO4) right-arrow GlcNSO4 ± (6-SO4) disaccharide units, interrupted by sulfate-poor (mainly N-acetylated) domains, with the relative proportions of these domains varying between different syndecans (33). Laminin-1 high affinity dodecasaccharide is a sulfate-rich oligosaccharide, containing repeating disaccharide units of IdoA(2-SO4) right-arrow GlcNSO4(6-SO4), and is derived from the cell surface of MCF-7 cells. Therefore, it is possible to conclude that this oligosaccharide is part of the sulfate-rich domains of syndecan HS. Syndecan I particularly, has been shown to bind to laminin-1 via heparan sulfate chains (34). However, even though MCF-7 cells synthesize heparan sulfate proteoglycans, it is unclear which of these syndecans carry the laminin-1-binding oligosaccharide in their HS chains.

Aside from their central role in tumor-host adhesion in metastasis, laminin-HS interactions may have biological importance in basement membrane assembly due to the ubiquitous occurrence of HS proteoglycans and laminin in basement membranes and the fact that laminin-1 contains putative heparin-binding sites (9). Perlecan, the predominant proteoglycan of basement membrane, is a HS proteoglycan and has been shown to bind to laminin-1 via its HS chains (35). In addition, Engelbreth-Holm-Swarm tumor heparan sulfate, most likely derived from perlecan, was shown to contain at least 80% of its sulfate residues in N-sulfated form (36). Furthermore, the disaccharide IdoA(2-SO4) right-arrow GlcNSO4(6-SO4) constitutes approximately 50% of the total disaccharide in Engelbreth-Holm-Swarm tumor heparan sulfate. It is possible that laminin-perlecan interactions could involve the laminin high affinity dodecasaccharide, composed of IdoA(2-SO4) right-arrow GlcNSO4(6-SO4) repeating disaccharide units, and the G domain of laminin-1. Such ionic interactions could contribute to the macromolecular assembly of basement membrane (37).

    FOOTNOTES

* This work was supported in part by grants from the North Carolina Biotechnology Center (to N. P.) and an intramural research grant (to N. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Section of Comparative Medicine, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, North Carolina 27157-1040. Tel.: 336-716-2699; Fax: 336-716-6279; E-mail: npartha{at}wfubmc.edu.

The abbreviations used are: HS, heparan sulfate; GMS, GluA right-arrow AManR(6-SO4)IMS, IdoA right-arrow AManR(6-SO4)ISM, IdoA(2-SO4) right-arrow AManRISMS, IdoA(2-SO4) right-arrow AManR(6-SO4)GSM, GluA(2-SO4) right-arrow AManRHPLC, high performance liquid chromatographybFGF, bovine fibroblast growth factorMEM, minimum essential medium.
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Abstract
Introduction
Procedures
Results
Discussion
References

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