A recombinant technique for mapping functional sites of heterotrimeric collagen helices: Collagen IV CB3 fragment as a prototype for integrin binding

Collagen superfamily of proteins is a major component of the extracellular matrix. Defects in collagens underlie the cause of nearly 40 human genetic diseases in millions of people worldwide. Pathogenesis typically involves genetic alterations of the triple helix, a hallmark structural feature that bestows exceptional mechanical resistance to tensile forces and a capacity to bind a plethora of macromolecules. Yet, there is a paramount knowledge gap in understanding the functionality of distinct sites along the triple helix. Here, we present a recombinant technique to produce triple helical fragments for functional studies. The experimental strategy utilizes the unique capacity of the NC2 heterotrimerization domain of collagen IX to drive three α-chain selection and registering the triple helix stagger. For proof of principle, we produced and characterized long triple helical fragments of collagen IV that were expressed in a mammalian system. The heterotrimeric fragments encompassed the CB3 trimeric peptide of collagen IV, which harbors the binding motifs for α1β1 and α2β1 integrins. Fragments were characterized and shown to have a stable triple helix, post-translational modifications, and high affinity and specific binding of integrins. The NC2 technique is a universal tool for the high-yield production of heterotrimeric fragments of collagens. Fragments are suitable for mapping functional sites, determining coding sequences of binding sites, elucidating pathogenicity and pathogenic mechanisms of genetic mutations, and production of fragments for protein replacement therapy. Collagen superfamily of proteins is a major component of the extracellular matrix. Defects in collagens underlie the cause of nearly 40 human genetic diseases in millions of people worldwide. Pathogenesis typically involves genetic alterations of the triple helix, a hallmark structural feature that bestows exceptional mechanical resistance to tensile forces and a capacity to bind a plethora of macromolecules. Yet, there is a paramount knowledge gap in understanding the functionality of distinct sites along the triple helix. Here, we present a recombinant technique to produce triple helical fragments for functional studies. The experimental strategy utilizes the unique capacity of the NC2 heterotrimerization domain of collagen IX to drive three α-chain selection and registering the triple helix stagger. For proof of principle, we produced and characterized long triple helical fragments of collagen IV that were expressed in a mammalian system. The heterotrimeric fragments encompassed the CB3 trimeric peptide of collagen IV, which harbors the binding motifs for α1β1 and α2β1 integrins. Fragments were characterized and shown to have a stable triple helix, post-translational modifications, and high affinity and specific binding of integrins. The NC2 technique is a universal tool for the high-yield production of heterotrimeric fragments of collagens. Fragments are suitable for mapping functional sites, determining coding sequences of binding sites, elucidating pathogenicity and pathogenic mechanisms of genetic mutations, and production of fragments for protein replacement therapy. Collagens are a major component of the extracellular matrix. They are comprised of 28 types in humans (I–XXVIII) encoded by over 40 different genes, forming a diversity of triple helical protomers of varying α-chain compositions (1Bächinger H.P. Mizuno K. Vranka J.A. Boudko S.P. Collagen formation and structure.in: Comprehensive Natural Products II: Chemistry and Biology. Elsevier Ltd, Kidlington2010: 469-530Crossref Google Scholar, 2Ricard-Blum S. The collagen family.Cold Spring Harb. Perspect. Biol. 2011; 3: a004978Crossref PubMed Scopus (1232) Google Scholar). Protomers assemble into diverse superstructures, ranging from networks to fibrils and broadly function in structural, mechanical, and organizational roles that define tissue architecture and influence cellular behavior. Defects in collagens underlie the cause of nearly 40 human genetic diseases, affecting numerous organs and tissues in millions of people worldwide (3Fidler A.L. Boudko S.P. Rokas A. Hudson B.G. The triple helix of collagens - an ancient protein structure that enabled animal multicellularity and tissue evolution.J. Cell Sci. 2018; 131Crossref PubMed Scopus (87) Google Scholar). Pathogenesis typically involves genetic alterations of the triple helix, a hallmark structural feature that bestows exceptional mechanical resistance to tensile forces and a capacity to bind a plethora of macromolecules. Such macromolecules include but are not limited to integrins, DDR1 and 2, fibronectin, nidogen, perlecan, heparin, von Willebrand factor, decorin, bone morphogenetic proteins, and glycoprotein VI (2Ricard-Blum S. The collagen family.Cold Spring Harb. Perspect. Biol. 2011; 3: a004978Crossref PubMed Scopus (1232) Google Scholar, 4Parkin J.D. San Antonio J.D. Pedchenko V. Hudson B. Jensen S.T. Savige J. Mapping structural landmarks, ligand binding sites, and missense mutations to the collagen IV heterotrimers predicts major functional domains, novel interactions, and variation in phenotypes in inherited diseases affecting basement membranes.Hum. Mutat. 2011; 32: 127-143Crossref PubMed Scopus (85) Google Scholar, 5San Antonio J.D. Jacenko O. Fertala A. Orgel J. Collagen structure-function mapping informs applications for regenerative medicine.Bioengineering (Basel). 2020; 8: 3Crossref PubMed Scopus (34) Google Scholar). Yet, there is a paramount knowledge gap in understanding the functionality of distinct sites along the triple helix. This lack of knowledge impedes the development of precision therapies aimed at restoring/repairing function of collagen superstructures. A unique feature of the collagen triple helix is a register (or stagger) of chains (6Okuyama K. Revisiting the molecular structure of collagen.Connect. Tissue Res. 2008; 49: 299-310Crossref PubMed Scopus (93) Google Scholar). There are always leading, middle, and trailing chains shifted by one residue. Even in homotrimeric types of collagens, identical residues within the triple helix are not structurally equivalent. At least seven types of human collagens, that is, I, IV, V, VI, VIII, IX, and XI, exist as heterotrimers of either AAB or ABC forms (2Ricard-Blum S. The collagen family.Cold Spring Harb. Perspect. Biol. 2011; 3: a004978Crossref PubMed Scopus (1232) Google Scholar), which represent an additional challenge in generating biologically relevant collagen fragments. Examples include collagen I, the most abundant fibrillar type with an α1α1α2 composition and collagen IV of basement membrane with three compositions in mammals—α1α1α2, α3α4α5, and α5α5α6 (7Khoshnoodi J. Pedchenko V. Hudson B.G. Mammalian collagen IV.Microsc. Res. Tech. 2008; 71: 357-370Crossref PubMed Scopus (485) Google Scholar, 8Hudson B.G. Reeders S.T. Tryggvason K. Type IV collagen: structure, gene organization, and role in human diseases. Molecular basis of Goodpasture and Alport syndromes and diffuse leiomyomatosis.J. Biol. Chem. 1993; 268: 26033-26036Abstract Full Text PDF PubMed Google Scholar). Generation of short fragments of homotrimeric collagens using chemical synthesis became a general method, and great progress was achieved using this approach. Synthetic peptides were used to solve the first crystal structures of a collagen triple helix (9Okuyama K. Okuyama K. Arnott S. Takayanagi M. Kakudo M. Crystal and molecular structure of a collagen-like polypeptide (Pro-Pro-Gly)10.J. Mol. Biol. 1981; 152: 427-443Crossref PubMed Scopus (168) Google Scholar), the collagen triple helix with a mutation (10Bella J. Eaton M. Brodsky B. Berman H.M. Crystal and molecular structure of a collagen-like peptide at 1.9 A resolution.Science. 1994; 266: 75-81Crossref PubMed Scopus (904) Google Scholar), and a collagen triple helix complex with integrin α2β1 (11Emsley J. Knight C.G. Farndale R.W. Barnes M.J. Liddington R.C. Structural basis of collagen recognition by integrin alpha2beta1.Cell. 2000; 101: 47-56Abstract Full Text Full Text PDF PubMed Scopus (854) Google Scholar). Peptide toolkits covering the entire triple-helical domains of homotrimeric collagens II and III were successfully implemented and used to precisely map and study binding to integrin α2β1 (12Raynal N. Hamaia S.W. Siljander P.R. Maddox B. Peachey A.R. Fernandez R. et al.Use of synthetic peptides to locate novel integrin alpha2beta1-binding motifs in human collagen III.J. Biol. Chem. 2006; 281: 3821-3831Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), von Willebrand factor (13Lisman T. Raynal N. Groeneveld D. Maddox B. Peachey A.R. Huizinga E.G. et al.A single high-affinity binding site for von Willebrand factor in collagen III, identified using synthetic triple-helical peptides.Blood. 2006; 108: 3753-3756Crossref PubMed Scopus (106) Google Scholar), DDR1 (14Xu H. Raynal N. Stathopoulos S. Myllyharju J. Farndale R.W. Leitinger B. Collagen binding specificity of the discoidin domain receptors: binding sites on collagens II and III and molecular determinants for collagen IV recognition by DDR1.Matrix Biol. 2011; 30: 16-26Crossref PubMed Scopus (140) Google Scholar), DDR2 (15Konitsiotis A.D. Raynal N. Bihan D. Hohenester E. Farndale R.W. Leitinger B. Characterization of high affinity binding motifs for the discoidin domain receptor DDR2 in collagen.J. Biol. Chem. 2008; 283: 6861-6868Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar), dermatopontin (16Jensen M.M. Bonna A. Frederiksen S.J. Hamaia S.W. Hojrup P. Farndale R.W. et al.Tyrosine-sulfated dermatopontin shares multiple binding sites and recognition determinants on triple-helical collagens with proteins implicated in cell adhesion and collagen folding, fibrillogenesis, cross-linking, and degradation.Biochim. Biophys. Acta Proteins Proteom. 2022; 1870140771Crossref PubMed Scopus (2) Google Scholar), HSP47 (17Cai H. Sasikumar P. Little G. Bihan D. Hamaia S.W. Zhou A. et al.Identification of HSP47 binding site on native collagen and its implications for the development of HSP47 inhibitors.Biomolecules. 2021; 11: 983Crossref PubMed Scopus (7) Google Scholar), gpVI (18Jarvis G.E. Raynal N. Langford J.P. Onley D.J. Andrews A. Smethurst P.A. et al.Identification of a major GpVI-binding locus in human type III collagen.Blood. 2008; 111: 4986-4996Crossref PubMed Scopus (61) Google Scholar), LAIR-1 (19Lebbink R.J. Raynal N. de Ruiter T. Bihan D.G. Farndale R.W. Meyaard L. Identification of multiple potent binding sites for human leukocyte associated Ig-like receptor LAIR on collagens II and III.Matrix Biol. 2009; 28: 202-210Crossref PubMed Scopus (81) Google Scholar), SPARC (20Giudici C. Raynal N. Wiedemann H. Cabral W.A. Marini J.C. Timpl R. et al.Mapping of SPARC/BM-40/osteonectin-binding sites on fibrillar collagens.J. Biol. Chem. 2008; 283: 19551-19560Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), MMP-3 (21Manka S.W. Bihan D. Farndale R.W. Structural studies of the MMP-3 interaction with triple-helical collagen introduce new roles for the enzyme in tissue remodelling.Sci. Rep. 2019; 918785Crossref Scopus (26) Google Scholar), multimerin 1 (22Leatherdale A. Parker D. Tasneem S. Wang Y. Bihan D. Bonna A. et al.Multimerin 1 supports platelet function in vivo and binds to specific GPAGPOGPX motifs in fibrillar collagens that enhance platelet adhesion.J. Thromb. Haemost. 2021; 19: 547-561Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar), and other proteins (reviewed by Farndale (23Farndale R.W. Collagen-binding proteins: insights from the collagen toolkits.Essays Biochem. 2019; 63: 337-348Crossref PubMed Scopus (15) Google Scholar)). Several strategies were developed to address production of heterotrimeric collagen fragments using regioselective chemical synthesis (24Sacca B. Moroder L. Synthesis of heterotrimeric collagen peptides containing the alpha1beta1 integrin recognition site of collagen type IV.J. Pept. Sci. 2002; 8: 192-204Crossref PubMed Scopus (31) Google Scholar, 25Ottl J. Battistuta R. Pieper M. Tschesche H. Bode W. Kuhn K. et al.Design and synthesis of heterotrimeric collagen peptides with a built-in cystine-knot. Models for collagen catabolism by matrix-metalloproteases.FEBS Lett. 1996; 398: 31-36Crossref PubMed Scopus (85) Google Scholar, 26Boulegue C. Musiol H.J. Gotz M.G. Renner C. Moroder L. Natural and artificial cystine knots for assembly of homo- and heterotrimeric collagen models.Antioxid. Redox Signal. 2008; 10: 113-125Crossref PubMed Scopus (21) Google Scholar) or hosting heterotrimers with artificial sequence designs (27Jalan A.A. Sammon D. Hartgerink J.D. Brear P. Stott K. Hamaia S.W. et al.Chain alignment of collagen I deciphered using computationally designed heterotrimers.Nat. Chem. Biol. 2020; 16: 423-429Crossref PubMed Scopus (20) Google Scholar, 28Acevedo-Jake A.M. Clements K.A. Hartgerink J.D. Synthetic, register-specific, AAB heterotrimers to investigate single point glycine mutations in osteogenesis imperfecta.Biomacromolecules. 2016; 17: 914-921Crossref PubMed Scopus (15) Google Scholar) (reviewed by Xu and Kirchner (29Xu Y. Kirchner M. Collagen mimetic peptides.Bioengineering (Basel). 2021; 8: 5Crossref PubMed Scopus (26) Google Scholar)). However, these approaches are limited to relatively short fragments (up to 12 residues) and require sophisticated approaches of chemical synthesis. So far, no collagen peptide toolkits have been reported for any heterotrimeric type of collagen. Here, we present a recombinant technique to produce heterotrimeric (triple helical) fragments for mapping functional sites, determining coding sequences of binding sites, elucidating pathogenicity and pathogenic mechanisms of genetic mutations, and production of fragments for protein replacement therapy. The experimental strategy utilizes the unique property of the noncollagenous (NC) 2 heterotrimerization domain of collagen IX. We previously demonstrated that the NC2 domain is sufficient to drive three α-chain selections and register the triple helix stagger using a bacterial system (30Boudko S.P. Bachinger H.P. The NC2 domain of type IX collagen determines the chain register of the triple helix.J. Biol. Chem. 2012; 287: 44536-44545Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 31Boudko S.P. Bachinger H.P. Structural insight for chain selection and stagger control in collagen.Sci. Rep. 2016; 637831Crossref PubMed Scopus (21) Google Scholar, 32Boudko S.P. Zientek K.D. Vance J. Hacker J.L. Engel J. Bachinger H.P. The NC2 domain of collagen IX provides chain selection and heterotrimerization.J. Biol. Chem. 2010; 285: 23721-23731Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). For proof of principle, we adapted the NC2 domain for studies of collagen IV (Fig. 1), which harbors binding sites for numerous macromolecules (3Fidler A.L. Boudko S.P. Rokas A. Hudson B.G. The triple helix of collagens - an ancient protein structure that enabled animal multicellularity and tissue evolution.J. Cell Sci. 2018; 131Crossref PubMed Scopus (87) Google Scholar, 4Parkin J.D. San Antonio J.D. Pedchenko V. Hudson B. Jensen S.T. Savige J. Mapping structural landmarks, ligand binding sites, and missense mutations to the collagen IV heterotrimers predicts major functional domains, novel interactions, and variation in phenotypes in inherited diseases affecting basement membranes.Hum. Mutat. 2011; 32: 127-143Crossref PubMed Scopus (85) Google Scholar). Long triple helical fragments that encompass the CB3 site that harbors the binding motifs for α1β1 and α2β1 integrins (Fig. 1) were expressed and characterized. Fragments were shown to have a stable triple helix, post-translational modifications (PTMs), and specific binding to integrins. Expression of artificial or fragmentary collagen triple helices that exhibit native structure and function has proven difficult as they are composed of two or three distinct α-chains. It has proven difficult to control the stoichiometry of the assembled collagen fragment. Moreover, collagen sequences require PTMs, that is, hydroxylation and glycosylation, in order to form a stable and functional triple helical structure. We thus sought an expression system in mammalian cells that would facilitate expression of collagen domains whose collagen subunit composition and helix register could be controlled. Previously, we demonstrated that three chains of the collagen IX NC2 domain, one for each subunit, are sufficient to drive subunit selection, that is, appropriate subunit composition and to control the register of the triple helical stagger (30Boudko S.P. Bachinger H.P. The NC2 domain of type IX collagen determines the chain register of the triple helix.J. Biol. Chem. 2012; 287: 44536-44545Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 31Boudko S.P. Bachinger H.P. Structural insight for chain selection and stagger control in collagen.Sci. Rep. 2016; 637831Crossref PubMed Scopus (21) Google Scholar, 32Boudko S.P. Zientek K.D. Vance J. Hacker J.L. Engel J. Bachinger H.P. The NC2 domain of collagen IX provides chain selection and heterotrimerization.J. Biol. Chem. 2010; 285: 23721-23731Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Here, we adapt this system to produce a long fragment of collagen IV (∼200 residues) in a mammalian expression system. We selected the CB3 region of human collagen IV as its most well-characterized fragment (33Vandenberg P. Kern A. Ries A. Luckenbill-Edds L. Mann K. Kuhn K. Characterization of a type IV collagen major cell binding site with affinity to the alpha 1 beta 1 and the alpha 2 beta 1 integrins.J. Cell Biol. 1991; 113: 1475-1483Crossref PubMed Scopus (206) Google Scholar, 34Underwood P.A. Bennett F.A. Kirkpatrick A. Bean P.A. Moss B.A. Evidence for the location of a binding sequence for the alpha 2 beta 1 integrin of endothelial cells, in the beta 1 subunit of laminin.Biochem. J. 1995; 309: 765-771Crossref PubMed Scopus (31) Google Scholar, 35Plaisier E. Chen Z. Gekeler F. Benhassine S. Dahan K. Marro B. et al.Novel COL4A1 mutations associated with HANAC syndrome: a role for the triple helical CB3[IV] domain.Am. J. Med. Genet. A. 2010; 152A: 2550-2555Crossref PubMed Scopus (80) Google Scholar, 36Kern A. Eble J. Golbik R. Kuhn K. Interaction of type IV collagen with the isolated integrins alpha 1 beta 1 and alpha 2 beta 1.Eur. J. Biochem. 1993; 215: 151-159Crossref PubMed Scopus (188) Google Scholar, 37Fleischmajer R. Perlish J.S. MacDonald 2nd, E.D. Schechter A. Murdoch A.D. Iozzo R.V. et al.There is binding of collagen IV to beta 1 integrin during early skin basement membrane assembly.Ann. N. Y. Acad. Sci. 1998; 857: 212-227Crossref PubMed Scopus (35) Google Scholar, 38Eble J.A. Ries A. Lichy A. Mann K. Stanton H. Gavrilovic J. et al.The recognition sites of the integrins alpha1beta1 and alpha2beta1 within collagen IV are protected against gelatinase A attack in the native protein.J. Biol. Chem. 1996; 271: 30964-30970Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 39Eble J.A. Golbik R. Mann K. Kuhn K. The alpha 1 beta 1 integrin recognition site of the basement membrane collagen molecule [alpha 1(IV)]2 alpha 2(IV).EMBO J. 1993; 12: 4795-4802Crossref PubMed Scopus (179) Google Scholar, 40Dinkla K. Talay S.R. Morgelin M. Graham R.M. Rohde M. Nitsche-Schmitz D.P. et al.Crucial role of the CB3-region of collagen IV in PARF-induced acute rheumatic fever.PLoS One. 2009; 4e4666Crossref PubMed Scopus (44) Google Scholar, 41Calderwood D.A. Tuckwell D.S. Eble J. Kuhn K. Humphries M.J. The integrin alpha1 A-domain is a ligand binding site for collagens and laminin.J. Biol. Chem. 1997; 272: 12311-12317Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The CB3 region of collagen IV was initially identified as a fragment chemically excised with cyanogen bromide (CNBr) that could be prepared in relatively high yield for structural and functional studies (33Vandenberg P. Kern A. Ries A. Luckenbill-Edds L. Mann K. Kuhn K. Characterization of a type IV collagen major cell binding site with affinity to the alpha 1 beta 1 and the alpha 2 beta 1 integrins.J. Cell Biol. 1991; 113: 1475-1483Crossref PubMed Scopus (206) Google Scholar). Typical of collagen IV domains, CB3 has interruptions and a cystine knot as well as containing other known protein-binding sites (4Parkin J.D. San Antonio J.D. Pedchenko V. Hudson B. Jensen S.T. Savige J. Mapping structural landmarks, ligand binding sites, and missense mutations to the collagen IV heterotrimers predicts major functional domains, novel interactions, and variation in phenotypes in inherited diseases affecting basement membranes.Hum. Mutat. 2011; 32: 127-143Crossref PubMed Scopus (85) Google Scholar). The cystine knot seems to be an evolutionary conserved structural element in collagen IV also found in such distant organisms as fruit flies, roundworms, and even in Cnidaria (Fig. S1). We generated expression constructs that extended the sequences of CB3 to a gelatinase A cleavage site at the N terminus (38Eble J.A. Ries A. Lichy A. Mann K. Stanton H. Gavrilovic J. et al.The recognition sites of the integrins alpha1beta1 and alpha2beta1 within collagen IV are protected against gelatinase A attack in the native protein.J. Biol. Chem. 1996; 271: 30964-30970Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) and completed the last GXY repeat in the α2 chain to ensure the continuity and stability of the triple helices (Figure 1, Figure 2, Figure 3 and S2). We refer to this genetically encoded CB3 domain as extended CB3 (eCB3).Figure 3The chains and eCB3 assemblies. Schematic presentation of primary sequences of chains coexpressed to assemble 111, 222, 112, 121, and 211 eCB3 fragments. Linear scale corresponds to number of residues. The signal peptide sequences are excluded. Triple helical sequences are depicted as bars, yellow color corresponds to α1 chain of human collagen IV, light purple corresponds to α2, white to artificial GPP repeats. The sequences for NC2 domain are shown as pink, light blue, and light green rectangles and labeled A, B, and C, which corresponds to α1, α2, and α3 chains of human collagen IX. Interruptions, linkers, and tags are depicted as black lines. Cysteines are depicted as red pins.View Large Image Figure ViewerDownload Hi-res image Download (PPT) As in the bacterial expression system (30Boudko S.P. Bachinger H.P. The NC2 domain of type IX collagen determines the chain register of the triple helix.J. Biol. Chem. 2012; 287: 44536-44545Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 32Boudko S.P. Zientek K.D. Vance J. Hacker J.L. Engel J. Bachinger H.P. The NC2 domain of collagen IX provides chain selection and heterotrimerization.J. Biol. Chem. 2010; 285: 23721-23731Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar), for each chain (α1 and α2) of eCB3, we generated three constructs, each containing one of the three nonidentical NC2 trimerization domains of collagen IX, that is, α1 NC2, α2 NC2, and α3 NC2. These domains pack in a specific orientation around the threefold pseudosymmetry axis of collagen and define the stagger of the collagen triple helices (31Boudko S.P. Bachinger H.P. Structural insight for chain selection and stagger control in collagen.Sci. Rep. 2016; 637831Crossref PubMed Scopus (21) Google Scholar). Each of the three eCB3 domain sequences was cloned upstream of its specific NC2 domain and flanked by GPP repeats to avoid destabilizing effects at the N and C termini (Figs. 2 and S2). These extensions may also contribute to more robust PTMs within the CB3 sequences. To ensure straightforward affinity purifications of heterotrimeric assemblies, each chain contained a specific tag placed at the N terminus following the signal peptide (Figure 2, Figure 3 and S2). We selected the expiCHO transient expression system (Thermo Fisher), which is suitable for our need of coexpression of three different chains (Table S3). The transient transfection system we used allows for rapid production as it eliminates three cycles of time-consuming selection for each vector to generate stable clones and the necessity of each vector having a unique selectable marker. To facilitate collagen-specific PTMs, the expression cell media were supplemented with fresh ascorbate on a daily basis (42Lunstrum G.P. Bachinger H.P. Fessler L.I. Duncan K.G. Nelson R.E. Fessler J.H. Drosophila basement membrane procollagen IV. I. protein characterization and distribution.J. Biol. Chem. 1988; 263: 18318-18327Abstract Full Text PDF PubMed Google Scholar, 43Murad S. Grove D. Lindberg K.A. Reynolds G. Sivarajah A. Pinnell S.R. Regulation of collagen synthesis by ascorbic acid.Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 2879-2882Crossref PubMed Scopus (360) Google Scholar). The chain register of the collagen triple helix has long been considered critical for structure, folding, and function. For collagen IV, the register was explored only for helix 9 (39Eble J.A. Golbik R. Mann K. Kuhn K. The alpha 1 beta 1 integrin recognition site of the basement membrane collagen molecule [alpha 1(IV)]2 alpha 2(IV).EMBO J. 1993; 12: 4795-4802Crossref PubMed Scopus (179) Google Scholar), and no rules were reported on how it can be translated through the interruptions to other helices. However, in collagen IX, the register of the collagen triple helix N-terminal to the NC2 domain has been defined and shown to be determined by linkage to the NC2 domain (30Boudko S.P. Bachinger H.P. The NC2 domain of type IX collagen determines the chain register of the triple helix.J. Biol. Chem. 2012; 287: 44536-44545Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 31Boudko S.P. Bachinger H.P. Structural insight for chain selection and stagger control in collagen.Sci. Rep. 2016; 637831Crossref PubMed Scopus (21) Google Scholar). This known register allows matching to the correct register of the collagen triple helix if known. As the register of the C-terminal helix (#11, Fig. 1) of eCB3 fragment is not known, we generated plasmids to match each possible register. We refer to an assembly nomenclature in which the His-tagged NC2-A subunit (IX α1) is listed first, the FLAG-tagged NC2-B subunit (IX α2) is listed second, and the Twin-Strep-tagged NC2-C subunit (IX α3) is listed third (Fig. 3). We thus generated 112, 121, and 211 collagen IV eCB3-expressing cells. As it was also not known whether homopolymers assembled and were stable, we also expressed 111 and 222 eCB3 fragments (Fig. 3). Secreted proteins were purified from conditioned cell culture media using three sequential rounds of chain-specific affinity purifications (Figs. S3 and 4A). This purification scheme ensures the heterotrimeric composition and high purity (Fig. 4B). We achieved yields of pure proteins ranging from 1.5 to 2.4 mg from 1 l of culture medium for 111, 112, 121, and 211 eCB3 assemblies and ∼12 mg for the 222 eCB3 protein. To determine whether the purified proteins are stabilized by interchain disulfide bonds (Figs. 1B and 3), the samples were electrophoresed under both nonreducing and reducing conditions. A single band corresponding to disulfide cross-linked trimer for each of the 111, 112, 121, and 211 eCB3 assemblies was observed at approximately 120 kDa (Fig. 4B). The same samples run under reducing conditions resolved in monomeric bands of approximately 40 kDa. This result suggests that register of the last eCB3 helix (#11, Fig. 1), which is deliberately controlled by the NC2 domain, is not critical for assembly. Another surprising observation of assembly of homotrimeric 111 raises a question whether a new isoform of collagen IV can exist. In contrast, the 222-expressing cells produced heterogeneously assembled material with both trimeric, dimeric, and a minority of monomeric-like material under nonreducing conditions. Under reducing conditions, a dominant band at the expected molecular weight for the monomers is observed along with bands of unknown compositions. Because the 222-eCB3 chain is likely poorly or inappropriately folded, we hypothesized it would be sensitive to proteolysis. Indeed, after 6 weeks of storage of purified material at 4 °C, only the 222-eCB3 chain demonstrated significant degradation (Fig. S4). The affinity-purified proteins were further analyzed by size-exclusion chromatography (Fig. 4C). The 111, 112, 121, and 211 eCB3 assemblies demonstrated a single major peak that corresponds to the expected hydrodynamic radius, whereas the major fraction of 222 was shifted to a smaller apparent size suggesting a more globular structure. PTM of collagenous sequence is critical for mediating the stability of the collagen helix and collagen function. Our expression system, while not derived from epithelialized cells, should perform the PTMs observed in vivo in collagen proteins. We noticed that the 111, 112, 121, and 211 assembly–containing proteins ran as relatively diffused bands on the SDS-PAGE gels. Moreover, the 222-eCB3 chains migrated faster than the other chains under nonreducing and reducing conditions (Fig. 4B) suggesting that this protein may not be efficiently post-translationally modified. Consistent with this hypothesis, the amount of secreted and purified 222 was 5 to 8 times higher, as judged from the SDS-PAGE (Fig. 4B) and gel filtration chromatography (Fig. 4C), possibly because of escaping the PTM machinery by yet unknown mechanism. The 112, 121, and 211 proteins differed from one another in reducing SDS-PAGE (Fig. 4B), suggesting that registration differences affected PTMs at chain level. Collectively,