Identification of Individual Alpha Chains of Type IV Collagen Non-Collagenous 1 Fraction

Introduction

The glomerular basement membrane (GBM), an important structural component of the renal filtration barrier, is disrupted in several kidney diseases, including Goodpasture (GP) Syndrome and Alport Syndrome. Within the GBM, type IV collagen forms two distinct heterotrimeric networks: the widely distributed α1/α2(IV) network and the specialized α3/α4/α5(IV) network (Figure 1)(1). The α3/α4/α5(IV) collagen network contributes to the development of autoimmune mechanisms implicated in GP syndrome and other glomerular pathologies. In particular, the α3 chain of non-collagenous 1 (NC1) domain acts as the primary autoantigen triggering immunologic reactions (2) .

Chondrex, Inc. provides NC1 domain fractions from rat, mouse, and bovine kidneys through enzymatic digestion with bacterial collagenase. The NC1 domains appear as monomers (~25 kDa) and dimers (~50 kDa) in SDS-PAGE analysis.  However, this result does not identify the individual chains of the NC1 domains.

To further characterize the composition of these NC1 fractions, Chondrex, Inc. employed a panel of monoclonal antibodies (mAbs), generated against human NC1 peptide sequences corresponding to the α1-α5 chains. This immunological approach enabled chain-specific detection, confirming the presence of α1–α5 NC1 domains in the NC1 fractions across species.

Figure 1. The α1/α2(IV) and α3/α4/α5(IV) collagen network structures in the kidney
 

Reagents

1. Monoclonal antibodies

Abbreviations: B: Bovine H: Human, Ms: Mouse, P: Porcine, R: Rat, NC1: Non-collagenous domain 1
 

 2. NC1 of type IV collagen

Assay protocol

1. NC1 Dissociation

To investigate antibody binding to different conformational states of NC1, a denaturing solution was employed. 6M guanidine was utilized to dissociate NC1 into its monomeric form prior to plate coating. This dissociation may increase mAb reactivity by exposing hidden or obscure epitopes located near hydrophobic regions when these surfaces become immobilized on the plate.

2. Enzyme-Linked Immunosorbent Assay (ELISA) Protocol

Microtiter plates were coated with 10 µg/ml of each NC1 fraction, prepared in either 0.05M phosphate-buffered saline (PBS, pH 7.4) or in 6M Guanidine/0.05M Tris-Buffered Saline (pH 7.5). The plates were then incubated overnight at 4°C.

Following incubation, plates were washed with PBS containing 0.05% Tween 20 (PBST) and then incubated for 2 hours at room temperature with anti-NC1 mAbs diluted in Solution C (Cat # 2072).  After washing with PBST, plates were incubated for 1 hour at room temperature with horseradish peroxidase (HRP)-conjugated anti-rat IgG antibody (Cat # 10214) also diluted in Solution C.

After a final PBST wash, the colorimetric reaction was developed by adding TMB (3,3',5,5'-tetramethylbenzidine) substrate (Cat # 90023) and incubating for 25 minutes at room temperature. The reaction was terminated with stop solution (Cat # 9016) and absorbance was measured at  450 nm, with 630 nm used as the reference wavelength.
 

Table 1. Reactivity of monoclonal antibodies against NC1 fraction from each species

Results

Dissociation of the NC1 fraction generally increased the reactivity of all α chains with mAbs recognizing linear amino acid sequences. Both rat and mouse NC1 fractions exhibited reactivity with chain-specific mAbs, indicating the presence of all five α chains (α1–α5). In contrast, the bovine NC1 fraction showed strong reactivity with α1- and α2-specific mAbs, but markedly reduced reactivity with α4- and α5-specific mAbs, and no detectable binding to the α3-specific mAb. Sequence alignment of the NC1 domain between bovine and human revealed amino acid differences near the C-terminal region which may affect epitope recognition (Table 2). In particular, mAb H31 likely recognizes an epitope within this divergent region, potentially explaining the lack of detection in the bovine α3 chain.

Moreover, the α3–α5 chains are known to assemble into heterotrimeric structures under native conditions. Because Chondrex, Inc.'s NC1 fractions are prepared in their native form, detection by any α3–α5-specific mAbs implies the presence of this trimeric complex. It is also plausible that the bovine NC1 fraction contains a relatively higher proportion of α1/α2 trimers compared to α3–α5 trimers, which could reduce mAb reactivity.
 

Conclusion

The NC1 fractions derived from mouse, rat, and bovine kidney tissues contain all five alpha chains (α1–α5), though their relative abundances may vary between batches. Despite interspecies differences in amino acid sequences, particularly at the C-terminal end, these NC1 antigens remain suitable for detecting anti-NC1 antibodies in serum samples from rodent models. Please refer to the related products/protocols below for more information.

 

Nephritis

Anti-Glomerular Basement Membrane (GBM) Monoclonal Antibodies

Mouse Immune Complex Glomerulonephritis Induced by Cationic BSA

Rat Anti-Glomerular Basement Membrane Nephritis Models
 

Alport Syndrome

Monoclonal Antibodies for Studying Alport Syndrome
 

References

1.    B. G. Hudson, K. Tryggvason, M. Sundaramoorthy, E. G. Neilson, Alport’s syndrome, Goodpasture’s syndrome, and type IV collagen. N. Engl. J. Med. 348, 2543–2556 (2003).

2.    The Goodpasture Autoantigen: Identification of Multiple Cryptic Epitopes on the NC1 Domain of the Α3 (IV) Collagen Chain.

Table 2. Alignment of amino acid sequence of each NC1 chain

Alpha 1
Positions    1-60  
Human : GFLVTRHSQTIDDPQCPSGTKILYHGYSLLYVQGNERAHGQDLGTAGSCLRKFSTMPFLF
Mouse : GFLVTRHSQTTDDPLCPPGTKILYHGYSLLYVQGNERAHGQDLGTAGSCLRKFSTMPFLF
Rat   : GFLVTRHSQTTDDPLCPPGTKILYHGYSLLYVQGNERAHGQDLGTAGSCLRKFSTMPFLF
Bovine: GFLVTRHSQTTDDPQCPPGTKILYHGYSLLYVQGNERAHGQDLGTAGSCLRKFSTMPFLF
       ********** *** ** ******************************************

Positions   61-120 
Human : CNINNVCNFASRNDYSYWLSTPEPMPMSMAPITGENIRPFISRCAVCEAPAMVMAVHSQT
Mouse : CNINNVCNFASRNDYSYWLSTPEPMPMSMAPISGDNIRPFISRCAVCEAPAMVMAVHSQT
Rat   : CNINNVCNFASRNDYSYWLSTPEPMPMSMAPISGDNIRPFISRCAVCEAPAMVMAVHSQT
Bovine: CNINNVCNFASRNDYSYWLSTPEPMPMSMAPITGENIRPFISRCAVCEAPAMVMAVHSQT
       ******************************** * *************************

Positions  121-180 
Human : IQIPPCPSGWSSLWIGYSFVMHTSAGAEGSGQALASPGSCLEEFRSAPFIECHGRGTCNY
Mouse : IQIPQCPNGWSSLWIGYSFVMHTSAGAEGSGQALASPGSCLEEFRSAPFIECHGRGTCNY
Rat   : IQIPQCPNGWSSLWIGYSFVMHTSAGAEGSGQALASPGSCLEEFRSAPFIECHGRGTCNY
Bovine: IQIPQCPTGWSSLWIGYSFVMHTSAGAEGSGQALASPGSCLEEFRSAPFIECHGRGTCNY
       **** ** ****************************************************

Positions  181-225 
Human : YANAYSFWLATIERSEMFKKPTPSTLKAGELRTHVSRCQVCMRRT
Mouse : YANAYSFWLATIERSEMFKKPTPSTLKAGELRTHVSRCQVCMRRT
Rat   : YANAYSFWLATIERSEMFKKPTPSTLKAGELRTHVSRCQVCMRRT
Bovine: YANAYSFWLATIERSEMFKKPTPSTLKAGELRTHVSRCQVCMRRT
       *********************************************

Alpha 2
Positions    1-60  
Human : GYLLVKHSQTDQEPMCPVGMNKLWSGYSLLYFEGQEKAHNQDLGLAGSCLARFSTMPFLY
Mouse : GYLLVKHSQTDQEPMCPVGMNKLWSGYSLLYFEGQEKAHNQDLGLAGSCLARFSTMPFLY
Rat   : GYLLVKHSQTDQEPMCPVGMNKLWSGYSLLYFEGQEKAHNQDLGLAGSCLARFSTMPFLY
Bovine: GYLLVKHSQTDKEPMCPVGMNKLWSGYSLLYFEGQEKAHNQDLGLAGSCLARFSTMPFLY
       *********** ************************************************

Positions   61-120 
Human : CNPGDVCYYASRNDKSYWLSTTAPLPMMPVAEDEIKPYISRCSVCEAPAIAIAVHSQDVS
Mouse : CNPGDVCYYASRNDKSYWLSTTAPLPMMPVAEEEIKPYISRCSVCEAPAVAIAVHSQDTS
Rat   : CNPGDVCYYASRNDKSYWLSTTAPLPMMPVAEEEIKPYISRCSVCEAPAVAIAVHSQDVS
Bovine: CNPGDVCYYASRNDKSYWLSTTAPLPMMPVAEEDIRPYISRCSVCEAPAVAIAVHSQDVS
       ********************************  * ************* ******** *

Positions  121-180 
Human : IPHCPAGWRSLWIGYSFLMHTAAGDEGGGQSLVSPGSCLEDFRATPFIECNGGRGTCHYY
Mouse : IPHCPAGWRSLWIGYSFLMHTAAGDEGGGQSLVSPGSCLEDFRATPFIECNGGRGTCHYF
Rat   : IPHCPAGWRSLWIGYSFLMHTAAGDEGGGQSLVSPGSCLEDFRATPFIECNGGRGTCHYF
Bovine: IPHCPAGWRSLWIGYSFLMHTAAGDEGGGQSLVSPGSCLEDFRATPFIECNGARGTCHYY
       **************************************************** ******

Positions  181-224 
Human : ANKYSFWLTTIPEQSFQGSPSADTLKAGLIRTHISRCQVCMKNL
Mouse : ANKYSFWLTTIPEQNFQSTPSADTLKAGLIRTHISRCQVCMKNL
Rat   : ANKYSFWLTTIPEQNFQSTPSADTLKAGLIRTHISRCQVCMKNL
Bovine: ANKYSFWLTTIPEQSFQGTPSADTLKAGLIRTHISRCQVCMKNB
      ************** **  ************************

Alpha 3
Positions    1-60  
Human : GFVFTRHSQTTAIPSCPEGTVPLYSGFSFLFVQGNQRAHGQDLGTLGSCLQRFTTMPFLF
Mouse : GFIFTRHSQTTAIPSCPEGTQPLYSGFSLLFVQGNKRAHGQDLGTLGSCLQRFTTMPFLF
Rat   : GFVFTRHSQTTAAPSCPEGTQPLYSGFSLLFVQGNERAHGQDLGTLGSCLQRFTTMPFLF
Bovine: GFVFTRHSQTTAIPSCPEGTEPLYSGFSLLFVQGNEQAHGQDLGTLGSCLQRFTTMPFLF
       ** ********* ******* ******* ******  ***********************

Positions   61-120 
Human : CNVNDVCNFASRNDYSYWLSTPALMPMNMAPITGRALEPYISRCTVCEGPAIAIAVHSQT
Mouse : CNINNVCNFASRNDYSYWLSTPALMPMDMAPISGRALEPYISRCTVCEGPAMAIAVHSQT
Rat   : CNVNRVCNFASRNDYSYWLSTPAPMPTDMAPITGKALEPYISRCTVCEGPAMVIAVHSQT
Bovine: CNINDVCNFASRNDYSYWLSTPAMIPMDMAPITGRALEPYISRCTVCEGPAIAIAVHSQT
       ** * ******************  *  **** * ****************  *******

Positions  121-180 
Human : TDIPPCPHGWISLWKGFSFIMFTSAGSEGTGQALASPGSCLEEFRASPFLECHGRGTCNY
Mouse : TAIPPCPQDWVSLWKGFSFIMFTSAGSEGAGQALASPGSCLEEFRASPFIECHGRGTCNY
Rat   : TTIPPCPHGWVSLWKGFSFIMFTGAGSEGAGQALASPGSCLEEFRANPFLECHGRGTCNY
Bovine: TDIPPCPAGWISLWKGFSFIMFTSAGSEGAGQALASPGSCLEEFRASPFIECHGRGTCNY
       * *****  * ************ ***** **************** ** **********

Positions  181-225 
Human : YSNSYSFWLASLNPERMFRKPIPSTVKAGELEKIISRCQVCMKKR
Mouse : YSNSYSFWLASLNPERMFRKPIPSTVKAGDLEKIISRCQVCMKKR
Rat   : YSNSYSFWLASLNPERMFRKPIPSTVKAGALENIISRCQVCMKER
Bovine: YSNSYSFWLASLDPKRMFRKPIPSTVKAGELENIISRCQVCMKMR
       ************ * ************** ** ********** *

Alpha 4
Positions    1-60  
Human : GFLLVLHSQTDQEPTCPLGMPRLWTGYSLLYLEGQEKAHNQDLGLAGSCLPVFSTLPFAY
Mouse : GFLLVLHSQTDQEPACPVGMPRLWTGYSLLYMEGQEKAHNQDLGLAGSCLPVFSTLPFAY
Rat   : GFLLVLHSQTDQEPACPVGMPRLWTGYSLLYMEGQEKAHNQDLGLAGSCLPMFSTLPFAY
Bovine: GFLLVLHSQTDGEPTCPMGMPRLWTGYSLLYLEGQERAHNQDLGLAGSCLPIFSTLPFAY
       *********** ** ** ************* **** ************** ********

Positions   61-120 
Human : CNIHQVCHYAQRNDRSYWLASAAPLPMMPLSEEAIRPYVSRCAVCEAPAQAVAVHSQDQS
Mouse : CNIHQVCHYAQRNDRSYWLSSAAPLPMMPLSEEEIRSYISRCAVCEAPAQAVAVHSQDQS
Rat   : CNIHQVCHYARRNDRSYWLASAAPLPMMPVSEEEIRPYISRCAVCEAPAQAVAVHSQDQS
Bovine: CNIHQVCHYARRNDRSYWLASTAPLPMTPLSEDEIRPYISRCAVCEAPAQAVAVHSQDQS
       ********** ******** * ***** * **  ** * *********************

Positions  121-180 
Human : IPPCPQTWRSLWIGYSFLMHTGAGDQGGGQALMSPGSCLEDFRAAPFLECQGRQGTCHFF
Mouse : IPPCPRTWRSLWIGYSFLMHTGAGDQGGGQALMSPGSCLEDFRAAPFVECQGRQGTCHFF
Rat   : IPPCPRAWRSLWIGYSFLMHTGAGDQGGGQALTSPGSCLEDFRAAPFLECQGRQGTCHFF
Bovine: IPPCPRAWRSLWIGYSFLMHTGAGDQGGGQALMSPGSCLEDFRAAPFLECQGRQGTCHFF
       *****  ************************* ************** ************

Positions  181-226 
Human : ANKYSFWLTTVKADLQFSSAPAPDTLKESQAQRQKISRCQVCVKYS
Mouse : ANEYSFWLTTVNPDLQFASGPSPDTLKEVQAQRRKISRCQVCMKHS
Rat   : ANEYSFWLTTVNPETQFTLAPSPDTLKEGQAQRQKISRCQVCVKYS
Bovine: ANKYSFWLTTVRPDLQFSSAPLPDTLKESHAQRQKISRCQVCVKHS
       ** ********    **   * ******  *** ******** * *

Alpha 5
Positions    1-60  
Human : GFLITRHSQTTDAPQCPQGTLQVYEGFSLLYVQGNKRAHGQDLGTAGSCLRRFSTMPFMF
Mouse : GFLITRHSQTTEAPQCPRGTVHIYEGFSLLYVQGNKRAHGQDLGTAGSCLRRFSTMPFMF
Rat   : GFLITRHSQTTDAPQCPQGTVQIYDGFSLLYVQGNKRAHGQDLGTAGSCLRRFSTMPFMF
Bovine: GFLITRHSQTTDAPQCPQGTIQVYEGFSLLYVQGNKRAHGQDLGTAGSCLRRFSTMPFMF
       *********** ***** **   * ***********************************

Positions   61-120 
Human : CNINNVCNFASRNDYSYWLSTPEPMPMSMQPLKGQSIQPFISRCAVCEAPAVVIAVHSQT
Mouse : CNINNVCNFASRNDYSYWLSTPEPMPMNMEPLKGQSIQPFISRCAVCEAPAVVIAVHSQT
Rat   : CNINNVCNFASRNDYSYWLSTPEPMPMSMEPLKGQSIQPFISRCSVCEAPAVVIAVHSQT
Bovine: CNINNVCNFASRNDYSYWLSTPEPMPMSMEPLKGQSIQPFISRCAVCEAPAVVIAVHSQT
       *************************** * ************** ***************

Positions  121-180 
Human : IQIPHCPQGWDSLWIGYSFMMHTSAGAEGSGQALASPGSCLEEFRSAPFIECHGRGTCNY
Mouse : IQIPHCPQGWDSLWIGYSFMMHTSAGAEGSGQALASPGSCLEEFRSAPFIECHGRGTCNY
Rat   : IQIPHCPQGWDSLWIGYSFMMHTSAGAEGSGQALASPGSCLEEFRSAPFIECHGRGTCNY
Bovine: IQIPRCPQGWDSLWIGYSFMMHTSAGAEGSGQALASPGSCLEEFRSAPFIECHGRGTCNY
       **** *******************************************************

Positions  181-225 
Human : YANSYSFWLATVDVSDMFSKPQSETLKAGDLRTRISRCQVCMKRT
Mouse : YANSYSFWLATVDMSDMFNKPQSETLKAGDLRTRISRCQVCMKRT
Rat   : YANSYSFWLATVDMPDMFSKPQSETLKAGDLRTRISRCQVCMKMT
Bovine: YANSYSFWLATVDVSDMFSKPQSETLKAGDLRTRISRCQVCMKRT
       *************  *** ************************ *

Note: Red/Blue sequences were used to develop each antibody
Red (H11, H22, H31, H41, H52)/ Blue (M54)