Heparan sulfate is necessary for the early formation of nascent fibronectin and collagen I fibrils at matrix assembly sites

Abstract

Fibronectin (FN), an essential component of the extracellular matrix (ECM), is assembled via a cell-mediated process in which integrin receptors bind secreted FN and mediate its polymerization into fibrils that extend between cells, ultimately forming an insoluble matrix. Our previous work using mutant Chinese hamster ovary (CHO) cells identified the glycosaminoglycan heparan sulfate (HS) and its binding to FN as essential for the formation of insoluble FN fibrils. In this study, we investigated the contributions of HS at an early stage of the assembly process using knockdown of exostosin-1 (EXT1), one of the glycosyltransferases required for HS chain synthesis. NIH 3T3 fibroblasts with decreased EXT1 expression exhibited a significant reduction in both FN and type I collagen in the insoluble matrix. We show that FN fibril formation is initiated at matrix assembly sites, and while these sites were formed by cells with EXT1 knockdown, their growth was stunted compared with wild-type cells. The most severe defect observed was in the polymerization of nascent FN fibrils, which was reduced 2.5-fold upon EXT1 knockdown. This defect was rescued by the addition of exogenous soluble heparin chains long enough to simultaneously bind multiple FN molecules. The activity of soluble heparin in this process indicates that nascent fibril formation depends on HS more so than on the protein component of a specific HS proteoglycan. Together, our results suggest that heparin or HS is necessary for concentrating and localizing FN molecules at sites of early fibril assembly. Fibronectin (FN), an essential component of the extracellular matrix (ECM), is assembled via a cell-mediated process in which integrin receptors bind secreted FN and mediate its polymerization into fibrils that extend between cells, ultimately forming an insoluble matrix. Our previous work using mutant Chinese hamster ovary (CHO) cells identified the glycosaminoglycan heparan sulfate (HS) and its binding to FN as essential for the formation of insoluble FN fibrils. In this study, we investigated the contributions of HS at an early stage of the assembly process using knockdown of exostosin-1 (EXT1), one of the glycosyltransferases required for HS chain synthesis. NIH 3T3 fibroblasts with decreased EXT1 expression exhibited a significant reduction in both FN and type I collagen in the insoluble matrix. We show that FN fibril formation is initiated at matrix assembly sites, and while these sites were formed by cells with EXT1 knockdown, their growth was stunted compared with wild-type cells. The most severe defect observed was in the polymerization of nascent FN fibrils, which was reduced 2.5-fold upon EXT1 knockdown. This defect was rescued by the addition of exogenous soluble heparin chains long enough to simultaneously bind multiple FN molecules. The activity of soluble heparin in this process indicates that nascent fibril formation depends on HS more so than on the protein component of a specific HS proteoglycan. Together, our results suggest that heparin or HS is necessary for concentrating and localizing FN molecules at sites of early fibril assembly. Assembly of a fibronectin (FN) extracellular matrix (ECM) is essential for embryogenesis and the development, repair, regeneration, and homeostasis of all tissues. Disrupted or disordered fibronectin assembly occurs in many diseases and is key to the progression of fibrosis and scarring. FN binding to integrin receptors, especially α5β1 integrin, is required for assembly (1Singh P. Carraher C. Schwarzbauer J.E. Assembly of fibronectin extracellular matrix.Annu. Rev. Cell Dev. Biol. 2010; 26: 397-419Crossref PubMed Scopus (544) Google Scholar, 2Maurer L.M. Ma W. Mosher D.F. Dynamic structure of plasma fibronectin.Crit. Rev. Biochem. Mol. 2015; 51: 213-227Crossref PubMed Scopus (55) Google Scholar). Fibril formation also depends on FN self-association, mediated by its N-terminal assembly domain (3Schwarzbauer J.E. Identification of the fibronectin sequences required for assembly of a fibrillar matrix.J. Cell Biol. 1991; 113: 1463-1473Crossref PubMed Scopus (177) Google Scholar). FN has many other binding partners, including the glycosaminoglycans (GAGs) heparan sulfate and heparin that interact with its main heparin-binding domain (HepII) (4Wilcox-Adelman S.A. Denhez F. Goetinck P.F. Syndecan-4 modulates focal adhesion kinase phosphorylation.J. Biol. Chem. 2002; 277: 32970-32977Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). A dose-dependent loss of FN matrix was detected when GAG chain addition to proteins was blocked with xylosides (5Galante L.L. Schwarzbauer J.E. Requirements for sulfate transport and the diastrophic dysplasia sulfate transporter in fibronectin matrix assembly.J. Cell Biol. 2007; 179: 999-1009Crossref PubMed Scopus (35) Google Scholar) further implicating GAGs in matrix assembly. Heparan sulfate (HS)-modified proteoglycans (HSPGs) are found within the ECM and at the cell surface. HS chains are elongated onto core proteins in the Golgi apparatus by a complex of glycosyltransferases, exostosin-1 (EXT1) and exostosin-2 (EXT2) (6Lind T. Tufaro F. McCormick C. Lindahl U. Lidholt K. The putative tumor suppressors EXT1 and EXT2 are glycosyltransferases required for the biosynthesis of heparan sulfate.J. Biol. Chem. 1998; 273: 26265-26268Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar, 7McCormick C. Leduc Y. Martindale D. Mattison K. Esford L.E. Dyer A.P. Tufaro F. The putative tumour suppressor EXT1 alters the expression of cell-surface heparan sulfate.Nat. Genet. 1998; 19: 158-161Crossref PubMed Scopus (320) Google Scholar, 8Busse M. Feta A. Presto J. Wilén M. Grønning M. Kjellén L. Kusche-Gullberg M. Contribution of EXT1, EXT2, and EXTL3 to heparan sulfate chain elongation.J. Biol. Chem. 2007; 282: 32802-32810Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 9Presto J. Thuveson M. Carlsson P. Busse M. Wilén M. Eriksson I. Kusche-Gullberg M. Kjellén L. Heparan sulfate biosynthesis enzymes EXT1 and EXT2 affect NDST1 expression and heparan sulfate sulfation.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 4751-4756Crossref PubMed Scopus (130) Google Scholar, 10Esko J.D. Selleck S.B. Order out of chaos: Assembly of ligand binding sites in heparan sulfate.Annu. Rev. Biochem. 2002; 71: 435-471Crossref PubMed Scopus (1203) Google Scholar). Screening of a mutagen-treated Chinese hamster ovary (CHO) cell library identified CHO-677 cells, which show significantly reduced EXT1 mRNA levels and GlcA/GlcNAc transferase activities compared with wild-type CHO cells. The mutation eliminates HS synthesis (11Wei G. Bai X. Gabb M.M. Bame K.J. Koshy T.I. Spear P.G. Esko J.D. Location of the glucuronosyltransferase domain in the heparan sulfate copolymerase EXT1 by analysis of Chinese hamster ovary cell mutants.J. Biol. Chem. 2000; 275: 27733-27740Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) and causes a severe deficiency in assembly of FN (12Chung C.Y. Erickson H.P. Glycosaminoglycans modulate fibronectin matrix assembly and are essential for matrix incorporation of tenascin-C.J. Cell Sci. 1997; 110: 1413-1419Crossref PubMed Google Scholar, 13Raitman I. Huang M.L. Williams S.A. Friedman B. Godula K. Schwarzbauer J.E. Heparin-fibronectin interactions in the development of extracellular matrix insolubility.Matrix Biol. 2018; 67: 107-122Crossref PubMed Scopus (14) Google Scholar) presumably due to decreased EXT1 expression although the specific mutation has not been determined. Mutations in EXT1 have been linked to skeletal abnormalities and osteochondromas (14Hameetman L. Szuhai K. Yavas A. Knijnenburg J. van Duin M. van Dekken H. Taminiau A.H. Cleton-Jansen A.M. Bovée J.V. Hogendoorn P.C. The role of EXT1 in nonhereditary osteochondroma: Identification of homozygous deletions.J. Natl. Cancer Inst. 2007; 99: 396-406Crossref PubMed Scopus (93) Google Scholar). Conditional knockout of EXT1 in limb bud mesenchyme causes defects in growth and differentiation of cartilage condensations, including an abnormal perichondrium with dispersed FN matrix (15Matsumoto Y. Matsumoto K. Irie F. Fukushi J. Stallcup W.B. Yamaguchi Y. Conditional ablation of the heparan sulfate-synthesizing enzyme Ext1 leads to dysregulation of bone morphogenic protein signaling and severe skeletal defects.J. Biol. Chem. 2010; 285: 19227-19234Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). It seems clear that HS plays a role in FN matrix assembly and organization both in vivo and in cell culture. Our previous work using CHO cells identified a role for HS in conversion of FN fibrils into a stabilized, detergent-insoluble form (13Raitman I. Huang M.L. Williams S.A. Friedman B. Godula K. Schwarzbauer J.E. Heparin-fibronectin interactions in the development of extracellular matrix insolubility.Matrix Biol. 2018; 67: 107-122Crossref PubMed Scopus (14) Google Scholar). Since insolubility is the final step in FN assembly, this finding shows that HS functions late in assembly. We also observed higher levels of detergent-soluble FN in lysates CHO cells with This that HS have role in assembly, in an HS FN in cells that and we knockdown of EXT1 in NIH 3T3 fibroblasts and investigated the of loss of HS on the progression of FN matrix assembly. We and matrix assembly sites sites of FN fibril nascent FN fibrils, and FN matrix in EXT1 knockdown we show that EXT1 mRNA expression in fibroblasts HS defects in the of FN the role of is chain We that in addition to its role in forming insoluble FN fibrils, HS binding to FN is also in early of assembly interactions by concentrating FN sites of matrix assembly the role of HS in assembly of an FN we NIH 3T3 which and levels of FN and into a fibrillar matrix. NIH 3T3 fibroblasts were with an the EXT1 of and shows compared with cells, EXT1 mRNA was significantly decreased with knockdown by at and at as by Since EXT1 mRNA levels to at all were within of FN levels were in and not show a significant In was significant in integrin levels between and EXT1 knockdown cells not in FN matrix assembly is not due to loss of FN or its EXT1 is required for synthesis of HS so we HS levels by HS was significantly for cell HS detected with an in EXT1 knockdown cells compared with cells A in was observed between wild-type CHO cells and CHO-677 cells results that knockdown of EXT1 significantly reduced HS decreased EXT1 and HS to a in FN matrix assembly, matrix levels were by at and cells assembled more FN matrix than EXT1 knockdown cells at all of FN matrix were significantly in EXT1 knockdown cells in to the cells FN fibrils are soluble in are ultimately to a which as a matrix for of other ECM proteins (1Singh P. Carraher C. Schwarzbauer J.E. Assembly of fibronectin extracellular matrix.Annu. Rev. Cell Dev. Biol. 2010; 26: 397-419Crossref PubMed Scopus (544) Google Scholar). of matrix a of matrix assembly. of FN higher FN levels in compared with EXT1 knockdown cells at and of FN to shows that FN levels in EXT1 knockdown cells the detected in cells at the that decreased matrix is not due to a in the of FN assembly. We that HS assembly of matrix in its assembly is In of this we found higher FN in the of EXT1 knockdown cells compared with cells while levels of FN are by in FN is assembled in the of The bone multiple is by in EXT1 and is by multiple K. EXT1 and EXT2 in multiple of Mol. PubMed Scopus Google Scholar). I the protein component of is for bone development and structure M. G. and of in the of type I collagen chains formation and in of PubMed Scopus Google Scholar). Since an FN matrix is necessary for collagen I of fibronectin in collagen to the domain of fibronectin both fibronectin and collagen organization in extracellular matrix.J. Cell Biol. PubMed Scopus Google Scholar, T. J. K. Mosher D.F. S. of type I and is on fibronectin and by and Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, J. F. I. J. J. collagen I modulates the cell to J. Cell 2007; PubMed Scopus Google Scholar, Schwarzbauer J.E. Fibronectin matrix as a for binding and collagen Biol. PubMed Scopus (14) Google we type I collagen levels were decreased in EXT1 knockdown fibroblasts via We a significant of to in type I collagen fibrils at and is secreted into the extracellular its and are by to collagen A. and as and Cell Biol. 2008; PubMed Scopus Google Scholar). of matrix collagen the most at and at and and or Schwarzbauer J.E. Fibronectin matrix as a for binding and collagen Biol. PubMed Scopus (14) Google Scholar). proteins were reduced in the EXT1 knockdown cells, collagen the most with an reduction at both show that EXT1 and HS are also required for formation of insoluble collagen fibrils. assembly reduced cell adhesion was by EXT1 knockdown. of and knockdown cells significant in a of FN We also cell on a FN and found that and knockdown cells cell the reduction in FN matrix with EXT1 knockdown is not due to defects in adhesion and The of EXT1 knockdown on fibril and insolubility our results with CHO-677 mutant cells (13Raitman I. Huang M.L. Williams S.A. Friedman B. Godula K. Schwarzbauer J.E. Heparin-fibronectin interactions in the development of extracellular matrix insolubility.Matrix Biol. 2018; 67: 107-122Crossref PubMed Scopus (14) Google Scholar) and further that HS is necessary for formation of insoluble FN matrix. It that HS also in the assembly process so we HS has a role in formation of matrix assembly sites, the sites of FN fibril assembly sites form FN when on FN binding sites for the N-terminal assembly domain of FN A. P. of fibronectin matrix assembly sites on fibroblasts and Cell Sci. 1997; 110: PubMed Google Scholar). N-terminal of which the assembly and were to matrix assembly sites by of cells was and the cell for was as a of matrix assembly both and knockdown cells sites of with significant in by binding to sites on cells was significantly higher than binding to knockdown cells by A and This finding shows that matrix assembly sites more in the of EXT1 and A of cells within the EXT1 knockdown higher levels of HS than the of the We this higher of HS with higher of matrix assembly HS of cells the and knockdown was and of matrix assembly sites and and the HS cell and the by and In the knockdown cells into using the and of the HS in the knockdown cells with HS within one of the cell at the were as knockdown cells the HS was than one the cell were as The HS by between these and The of knockdown cells that of cells at both and in and the cells significantly than the cells in the at both and The in between the and not to that the growth of matrix assembly sites in the is not with the the loss of HS is a severe deficiency in matrix assembly formation than their results our that matrix assembly development depends on is a of FN to the cell of matrix assembly, FN binding to and EXT1 knockdown fibroblasts was using cells to a that not matrix assembly and using cells in significant in FN binding was detected in not We that cell of FN by HS depends on the of matrix assembly matrix assembly sites into fibrillar and nascent fibrils that are ultimately into an insoluble fibrillar matrix Schwarzbauer J.E. Fibronectin fibril is upon of extracellular matrix Biol. PubMed Scopus Google Scholar). We in that matrix assembly formation by NIH 3T3 cells with exogenous FN and of FN fibrils were by FN and of A and show nascent fibrils in cells by both and and heparin nascent fibril assembly a to a to with heparin sulfate or was not to for heparin in nascent fibril formation nascent FN fibril assembly was significantly reduced in EXT1 knockdown fibroblasts and this deficiency was rescued by with or levels of nascent fibril assembly between knockdown and cells. HS is required for nascent FN fibril assembly and exogenous heparin this in cells that with type I collagen was significantly in NIH 3T3 cells with heparin in cell of cells in the of heparin assembled nascent collagen fibrils, which were not detected in the fibrils in both and with FN fibrils. that heparin of FN fibril assembly also type I collagen fibril The heparin to the EXT1 deficiency in fibril formation is to long bind FN simultaneously (11Wei G. Bai X. Gabb M.M. Bame K.J. Koshy T.I. Spear P.G. Esko J.D. Location of the glucuronosyltransferase domain in the heparan sulfate copolymerase EXT1 by analysis of Chinese hamster ovary cell mutants.J. Biol. Chem. 2000; 275: 27733-27740Abstract Full Text Full Text PDF PubMed Scopus (69) Google to and FN molecules. this is heparin chains that bind in nascent fibril assembly. We a heparin and with to bind one or FN (13Raitman I. Huang M.L. Williams S.A. Friedman B. Godula K. Schwarzbauer J.E. Heparin-fibronectin interactions in the development of extracellular matrix insolubility.Matrix Biol. 2018; 67: 107-122Crossref PubMed Scopus (14) Google Scholar, A. of heparan for specific of the heparin-binding domain of plasma fibronectin J. PubMed Scopus Google significant in nascent FN fibril was detected on cells with compared with NIH 3T3 cells A and was not to nascent fibril formation by EXT1 knockdown fibroblasts and significant were detected between with and in these not The heparin results show that binding between heparin and one or FN is not to nascent fibril assembly. We that more than FN molecules by a heparin chain to a in nascent fibrils. The domain of the has been in matrix assembly, and our previous with CHO-677 cells that HS a role for this GAG in formation of fibrils, a late stage in the assembly process (13Raitman I. Huang M.L. Williams S.A. Friedman B. Godula K. Schwarzbauer J.E. Heparin-fibronectin interactions in the development of extracellular matrix insolubility.Matrix Biol. 2018; 67: 107-122Crossref PubMed Scopus (14) Google Scholar). CHO cells not FN a matrix with exogenous FN Y. Schwarzbauer J.E. of fibronectin matrix assembly by of the type Cell Biol. PubMed Scopus Google Scholar). we show that NIH 3T3 fibroblasts that a FN matrix are on The of the of FN matrix fibrils, and the formation of matrix were all significantly decreased with knockdown of EXT1, one of the enzymes required to I collagen was also of HS at the in assembly identified a deficiency in formation of matrix assembly sites and nascent fibrils. matrix assembly sites were their growth was stunted in the of a role for HS in FN binding at these formation of nascent FN fibrils was also HS is the component was by of nascent fibril assembly with exogenous which was was long to bind multiple FN molecules. these we that HS has an early and role in matrix formation by FN molecules matrix assembly sites to nascent fibril assembly. heparin was to nascent fibril formation by EXT1 knockdown cells that HS not have to in to in these early of assembly. The on heparin chain binding of to FN not FN A heparin chain binding multiple FN molecules simultaneously as a to in interactions and (13Raitman I. Huang M.L. Williams S.A. Friedman B. Godula K. Schwarzbauer J.E. Heparin-fibronectin interactions in the development of extracellular matrix insolubility.Matrix Biol. 2018; 67: 107-122Crossref PubMed Scopus (14) Google Scholar). HS proteoglycans have been to by growth into of their receptors S. Esko J.D. Heparan sulfate Biol. PubMed Scopus Google and secreted proteins HS as a to D. Esko J.D. heparan Rev. Biochem. PubMed Scopus Google Scholar). HS also proteins to and the of and growth development D. Esko J.D. heparan Rev. Biochem. PubMed Scopus Google Scholar). A previous has that the and of the HS chain proteins to within the while to the cell the of signaling proteins as growth L. A. B. of growth in the matrix is by the of its binding sites in heparan Biol. PubMed Scopus Google Scholar). This also to of FN assembly by FN molecules to the cell and are at integrin of matrix assembly The in FN at these sites The that is required for assembly at the cell is by our finding that heparin not binding to cells in the of soluble heparin to shows that this is of affect FN on exogenous heparin FN matrix assembly by cells (5Galante L.L. Schwarzbauer J.E. Requirements for sulfate transport and the diastrophic dysplasia sulfate transporter in fibronectin matrix assembly.J. Cell Biol. 2007; 179: 999-1009Crossref PubMed Scopus (35) Google which FN than NIH 3T3 the of heparin on the of the of FN for binding that matrix assembly is by the of synthesis of both HS and in EXT1 and EXT2, the enzymes for HS an that is by osteochondromas G. G. G. A. multiple A of and Google Scholar). I is necessary for bone development M. G. and of in the of type I collagen chains formation and in of PubMed Scopus Google and show abnormal collagen and L. A. C. A. J. L. mutation abnormalities and collagen expression in the 2000; PubMed Scopus Google Scholar). type I collagen was significantly and with FN in cells and was reduced in EXT1 knockdown cells, which is with that FN a for type I collagen assembly and Schwarzbauer J.E. Fibronectin matrix as a for binding and collagen Biol. PubMed Scopus (14) Google Scholar). It is that defects in FN matrix by reduced or HS in a role in the early of to and other bone and are to bind to FN and in many cell activities (1Singh P. Carraher C. Schwarzbauer J.E. Assembly of fibronectin extracellular matrix.Annu. Rev. Cell Dev. Biol. 2010; 26: 397-419Crossref PubMed Scopus (544) Google Scholar, S. Esko J.D. Heparan sulfate Biol. PubMed Scopus Google Scholar). The of soluble heparin on matrix assembly not in this process the that to assembly on the and have in cell binding to FN A. of extracellular matrix assembly by Cell Sci. 2000; 113: PubMed Google Scholar, S. F. Denhez F. Mosher D.F. Goetinck P.F. Syndecan-4 with in a in the assembly of focal and Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The extracellular also with FN (12Chung C.Y. Erickson H.P. Glycosaminoglycans modulate fibronectin matrix assembly and are essential for matrix incorporation of tenascin-C.J. Cell Sci. 1997; 110: 1413-1419Crossref PubMed Google Scholar) and is by NIH 3T3 fibroblasts HS chains on FN with formed matrix assembly is a in in the matrix of osteochondromas and multiple with exostosin-1 (EXT1) 2008; 19: PubMed Scopus Google a between and the of specific in assembly, our a more of on FN matrix assembly. these we that the of matrix assembly by localizing and FN at nascent matrix assembly sites, its to the assembly process We not in of focal adhesion proteins or with or heparin M. that heparin of integrin binding multiple FN molecules a of this early of matrix assembly and nascent fibril HS the assembly of a FN matrix and collagen our also that on FN matrix assembly our EXT1 knockdown We that in the or of HS chains affect ECM assembly by the of matrix assembly FN and the of ECM We for this in previous work our which shows that reduced of proteoglycans with knockdown of diastrophic dysplasia sulfate transporter FN matrix assembly (5Galante L.L. Schwarzbauer J.E. Requirements for sulfate transport and the diastrophic dysplasia sulfate transporter in fibronectin matrix assembly.J. Cell Biol. 2007; 179: 999-1009Crossref PubMed Scopus (35) Google Scholar). Mutations in a of skeletal A. A. Mutations in the diastrophic dysplasia sulfate transporter mutation skeletal and PubMed Scopus Google due to loss of a FN matrix. both and as of the HS we our a for FN matrix assembly. NIH 3T3 fibroblasts were in with The cells were for Fibronectin was plasma or plasma via as Schwarzbauer J.E. The to the incorporation of plasma and into Cell Biol. PubMed Scopus Google Scholar). was cells with a the N-terminal of FN McCormick Schwarzbauer J.E. Fibronectin is mediated by sites within the of the Biol. Chem. Full Text PDF PubMed Google Scholar) and with was heparin was in this were Schwarzbauer J.E. Fibronectin matrix as a for binding and collagen Biol. PubMed Scopus (14) Google FN U. I and sulfate was to protein on were and and NIH 3T3 cells were at cells in a in cells were with EXT1 and in cells were with in at of was and cells were for at The was and with were and for all and EXT1 knockdown cells was to with by with a M. Schwarzbauer J.E. alters expression and in Biol. PubMed Scopus Google Scholar). was of using was with of The were in in a were as at and at for one at at and at for was for analysis was using EXT1 EXT1 FN FN analysis of matrix assembly, NIH 3T3 cells were at cells in a and for to for type I collagen analysis were with in the to collagen and were in and and were as I. Y. Schwarzbauer J.E. of fibronectin matrix Cell Biol. PubMed Google Scholar). were on or with and to were in A were at the I and were in A. were with development, was on with in the were to matrix cells were in a on in with or heparin as collagen cells were in with to collagen assembly in the FN heparin was also or at were and for matrix fibrils as Y. Schwarzbauer J.E. of fibronectin matrix assembly by of the type Cell Biol. PubMed Scopus Google Scholar). HS or knockdown NIH 3T3 cells, and CHO-677 cells were and were at the and I and were at and were were in were using a with a and using were in of for using were for using cells were in a on with a plasma FN in with the of analysis of nascent FN fibrils, plasma FN was to the cells were and with and FN fibril the the FN was in and the of the The FN fibril was by the of within the to for in cell The FN fibril cell of of was for and the of is analysis of matrix assembly sites, cell and the was with which was detected with were also with and and were in HS and a was cells using the and the within that was the within the was the was at cells and the of was are as the for a of were using an with are within the This The that have of with the of this We are to of the Schwarzbauer for their K. B. M. and J. S. K. B. M. and J. S. J. S. K. H. and B. M. L. K. B. M. and J. S. K. B. M. and J. S. 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