L-C1qDC-1, a novel C1q domain-containing protein from Lethenteron camtschaticum that is involved in the immune response

Accepted Manuscript L-C1qDC-1, a novel C1q domain-containing protein from Lethenteron camtschaticum that is involved in the immune response Guangying Pei, Ge Liu, Xiong Pan, Yue Pang, Qingwei Li PII:

S0145-305X(15)30033-1

DOI:

10.1016/j.dci.2015.08.011

Reference:

DCI 2450

To appear in:

Developmental and Comparative Immunology

Received Date: 12 May 2015 Revised Date:

21 August 2015

Accepted Date: 22 August 2015

Please cite this article as: Pei, G., Liu, G., Pan, X., Pang, Y., Li, Q., L-C1qDC-1, a novel C1q domaincontaining protein from Lethenteron camtschaticum that is involved in the immune response, Developmental and Comparative Immunology (2015), doi: 10.1016/j.dci.2015.08.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

L-C1qDC-1, a novel C1q domain-containing protein from

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Lethenteron camtschaticum that is involved in the immune response Guangying Pei1,2, Ge Liu1,2, Xiong Pan1,2, Yue Pang1,2*, Qingwei Li1,2*

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1

, College of Life Science, Liaoning Normal University, Dalian 116081, China

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2

, Lamprey Research Center, Liaoning Normal University, Dalian 116081, China

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* Corresponding author: Qingwei Li, Yue Pang

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Telephone and Fax: 0086-411-85827799.

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E-mail: [email protected]; [email protected]

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25 Pages, 6 Figures, 1 Supplementary Figures, 0 Supplementary Tables

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Abbreviations:

gC1q, globular C1q; L.camtschaticum, Lethenteron camtschaticum; C1qDC, C1q

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domain-containing; ghC1q, globular head C1q; VLRs, variable lymphocyte receptors;

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MBL, mannose-binding lectin; MASPs, MBL-associated serine proteases; FRP,

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fiber-reinforced plastic; BLASTx, Basic Local Alignment Search Tool X; NCBI,

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National Center for Biotechnology Information; NJ, neighbor-joining; ORF, open

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reading frame; RACE, rapid amplification of cDNA ends; IPTG, isopropyl

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β-D-thiogalactopyranoside; BCA, bicinchoninic acid; CFA, complete Freund’s

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adjuvant;

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3-aminopropyltriethoxy

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chemiluminescence; DAPI, 4’,6-diamidino-2-phenylindole; CCD, charge-coupled

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device; UTR, untranslated region; ELISA, enzyme-linked immunosorbent assay; TNF,

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GAPDH,

glyceraldehyde3-phosphate silane;

DAB,

1

dehydrogenase;

diaminobenzidine;

ECL,

APES, enhanced

ACCEPTED MANUSCRIPT 22

tumor necrosis factor.

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Abstract: The C1q domain-containing (C1qDC) proteins are a family of proteins

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characterized by a globular C1q (gC1q) domain at their C-terminus. These proteins

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are involved in various processes in vertebrates and are assumed to serve as important

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pattern recognition receptors in innate immunity in invertebrates. Here, a novel

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C1qDC protein from Lethenteron camtschaticum was identified and characterized

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(designated as L-C1qDC-1). After a partial cDNA sequence of L-C1qDC-1 was

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identified in a L.camtschaticum liver cDNA library, the full-length cDNA was

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obtained using 3’- and 5’-rapid amplification of cDNA ends (RACE). L-C1qDC-1

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encodes 236 amino acids and contains a signal peptide, a collagen-like sequence with

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Gly-Xaa-Yaa repeats, and a C-terminal gC1q domain. The L-C1qDC-1 protein was

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primarily distributed in the gut, liver and supraneural body of L.camtschaticum and

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was

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immunofluorescence

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immunofluorescence results showed that in L. camtschaticum serum, L-C1qDC-1

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could interact with variable lymphocyte receptor (VLR) B and displayed strong

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colocalization with cancer cell immune responses. These results indicated that the

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L-C1qDC-1 gene encodes a novel C1qDC protein that may play an important role in

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the immune responses of L.camtschaticum, providing clues for understanding the

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universal functions of C1qDC proteins in other species and suggesting that these

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proteins could serve as pattern recognition molecules in immunotherapy.

marginally

detectable

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leukocytes

Furthermore,

both

via

real-time

PCR

and

immunoprecipitation

and

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assays.

in

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Keywords: Lethenteron camtschaticum; L-C1qDC-1 protein; immune response

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1. Introduction C1q, the initiating subcomponent of the classical pathway of the complement

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system, is a versatile immune molecule. This molecule participates in an array of

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immune responses that are dependent on its globular C1q (gC1q) domain, which can

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bind a variety of self and non-self ligands (Färber et al., 2009; Kishore et al., 2004).

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As the key link connecting innate and adaptive immunity, C1q has the ability to bind

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immune complexes. This binding triggers activation of the classical complement

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pathway and induces a wide variety of immune responses, such as pathogen

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recognition (Matsushita et al., 2004), activation of the complement system (Kishore

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and Reid, 2000), phagocytosis of bacteria (Tahtouh et al., 2009), and mediation of cell

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migration (Bohlson et al., 2007). In addition, the broad-spectrum ligand-binding

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potential of C1q fuels its functional flexibility. C1q also engages in an array of

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processes that are completely independent of complement activation (Nayak et al.,

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2010), as is evident in the case of microglial activation in the central nervous system

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and maintenance of immune tolerance via clearance of apoptotic cells.

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In recent years, C1q domain-containing (C1qDC) proteins have been defined as a

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family that consists of all proteins containing a gC1q domain at their C-terminus

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(Carland and Gerwick, 2010; Ghai et al., 2007). More than 30 proteins have been

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identified as members of this family. The domain organization of these proteins

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primarily includes a leading signal peptide, a collagen-like region, and a gC1q domain.

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ACCEPTED MANUSCRIPT Based on their structural characteristics, C1qDC proteins are classified as

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C1q/C1q-like proteins containing a collagen region at the N-terminus or globular head

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C1q (ghC1q) proteins without a collagen region (Wang et al., 2012). These C1q

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proteins are common among nearly all vertebrates. Interestingly, agnathan lampreys,

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whose innate immune system is dissimilar to that of mammals, possess orthologs of

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C1q.

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Lampreys are the surviving representatives of jawless vertebrates, which are

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believed to have appeared approximately 500 million years ago (Osório and Rétaux,

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2008). Because of their unique position in the chordate phylogeny, lampreys are a key

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species for studying the evolutionary origin of the immune system (Amemiya et al.,

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2007; Han et al., 2008). In earlier studies, immunologists struggled to study the

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molecular mechanisms of the lamprey adaptive immune system, which is based on

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variable lymphocyte receptors (VLRs) (Han et al., 2008). Thus, lampreys may be the

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best model organism for research on the only known adaptive immune system not

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based on immunoglobulin (Ig) molecules. However, the innate immune system of

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lampreys presents great complexity. Although certain key complement components

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are present, such as the C1q-like proteins, mannose-binding lectin (MBL),

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MBL-associated serine proteases (MASP), Bf and C3, it remains unclear whether

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lampreys have a complement pathway.

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In this study, we identified a novel C1q family member (designated L-C1qDC-1) in

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Lethenteron camtschaticum, which differs from the C1q protein previously identified

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in lampreys that acts as a lectin (Matsushita et al., 2004). Additionally, we revealed 4

ACCEPTED MANUSCRIPT that an interaction between L-C1qDC-1 and VLRB in L.camtschaticum plays a

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prevalent role in L.camtschaticum immune responses.

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2. Materials and Methods

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2.1. Animals

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In December 2014, twenty adult L.camtschaticum specimens (length: 48-60 cm,

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weight: 112-274 g) were collected from the Songhua River in Heilongjiang Province,

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China, and were kept in fiber-reinforced plastic (FRP) tanks with running freshwater

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at Liaoning Normal University. Additionally, two New Zealand rabbits were obtained

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from Dalian Medical University, and six BALB/c mice were obtained from Beijing

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Kwinbon Biotechnology Co.LTD.

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2.2. Cloning of the full-length cDNA of the L-C1qDC-1 gene

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A C1qDC homolog from L.camtschaticum was obtained through analysis of a liver

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EST library using the Basic Local Alignment Search Tool X (BLASTx) of the

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National Center for Biotechnology Information (NCBI). Total RNA was extracted

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from L.camtschaticum livers using the Catrimox-14™ RNA Isolation Kit (TaKaRa,

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Japan) and then converted to cDNA with reverse transcriptase (Promega, USA). The

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full-length cDNA was amplified using a 3’-RACE (rapid amplification of cDNA ends)

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or 5’-RACE Core Set Kit with the 3’- and 5’-RACE primers shown in Table 1. The

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PCR product was purified and cloned into the pMD19-T vector using a DNA ligation

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kit (TaKaRa, Japan) prior to DNA sequencing.

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2.3. Amino acid sequence and phylogenetic analyses

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ACCEPTED MANUSCRIPT The amino acid sequence deduced from the L.camtschaticum L-C1qDC-1 gene was

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analyzed using online tools available at http://www.expasy.org/tools/scanprosite. Total

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amino acid sequence alignments of C1qDC family members, including L-C1qDC-1,

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were performed using ClustalX 1.81 with the default settings. The obtained results

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were converted into MEGA format and imported into MEGA 3.1 to construct a

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phylogenetic tree using the neighbor-joining (NJ) method and 1,000 bootstrapped

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replicates. Homology analysis of L-C1qDC-1 and C1q-like sequences was performed

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using BLASTx from NCBI. Glycosylation site and functional domain analyses of

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L-C1qDC-1

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http://www.cbs.dtu.dk/sevices/NetOGlyc/ and http://smart.embl-heidelberg.de/.

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2.4. Expression and purification of the rL-C1qDC-1 protein

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conducted

using

online

tools

at

The open reading frame (ORF) of L-C1qDC-1, flanked by EcoRI and HindIII

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restriction sites, was amplified and subcloned into the pCold I vector with a histidine

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(His) tag. Escherichia coli (E. coli) RosettaBlue (DE3) containing the pCold I

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-rL-C1qDC-1 plasmid was then cultured in 0.5 L of LB broth containing 30

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µg/mL kanamycin (Sangon Biotech, China) at 37°C for 3 h. When the OD600 reached

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0.7, isopropyl-β-D-thiogalactopyranoside (IPTG) (Sangon Biotech, China) was added

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to a final concentration of 1 mmol/L to induce protein expression. After 24 h of

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culture with shaking, cells were harvested via centrifugation at 6,000 rpm for 10 min

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at 4°C. The cell pellets were then resuspended, and the supernatants were purified to

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obtain the rL-C1qDC-1 protein under previously described conditions (Pang et al.,

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2015). Protein concentrations were estimated via the Bradford method using a

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ACCEPTED MANUSCRIPT Bicinchoninic Acid (BCA) Protein Assay Kit (Beyotime, China). The purified

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rL-C1qDC protein was then analyzed by 12% SDS-PAGE and stained with

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Coomassie Brilliant Blue (Sangon Biotech, China).

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2.5. Production, purification and specificity of anti-rL-C1qDC-1 antibodies (Abs)

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and western blotting

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Two New Zealand white rabbits were subcutaneously injected with purified

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rL-C1qDC-1 protein (400 µg/rabbit) mixed with complete Freund’s adjuvant (CFA)

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(Sigma, USA) at a 1:1 ratio, as described (Pang et al., 2012). For determination of the

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antiserum titers via enzyme-linked immunosorbent assay (ELISA), 380 ng of the

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rL-C1qDC-1 protein was diluted in 100 µL of polyclonal anti-rL-C1qDC-1 Ab at

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different dilutions (1,000-fold to 512,000-fold) in each well of a 96-well ELISA plate.

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For western blotting, total serum proteins were extracted from L.camtschaticum

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(Beyotime, China), and protein concentrations were determined using a BCA Protein

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Assay Kit (Beyotime, China). A total of 10 µg of purified rL-C1qDC-1 and 100 µg of

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native L-C1qDC-1 protein from different L.camtschaticum tissues were then

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subjected to 12% SDS-PAGE and transferred to nitrocellulose (NC) membranes.

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These membranes were blocked with 5% skim milk and incubated with rabbit

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anti-rL-C1qDC-1 Ab (1,000-fold dilution) overnight at 4°C, followed by incubation

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with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (5,000-fold

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dilution). The membranes were developed using an enhanced chemiluminescence

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(ECL) substrate (Beyotime, China). Of the two batches of polyclonal Abs from the

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two rabbits, we chose the batch from the rabbit with the higher titer for the subsequent

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ACCEPTED MANUSCRIPT experiments. To generate mouse anti-rL-C1qDC-1 monoclonal Abs (mAbs), BALB/c

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mice were immunized with the recombinant L-C1qDC-1. After four immunizations,

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the spleen cells of an immunized mouse were fused with SP2/0 myeloma cells.

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Culture medium containing hypoxanthine, aminopterin, and thymidine was used for

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screening of the fused hybridoma cells. Fourteen days after the cellfusion, the parent

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cells were diluted to 100 cells in 10 mL of mediumand cultured in a 96-well cell

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culture microplate (0.1 mL per well). The Abs secreted by the different clones were

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then assayed for their ability tobind to the antigen (Ag) using ELISA and an

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immunodot blot. The most productive and stable clone was selected for future use. To

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obtain a large number of Abs, hybridoma cells were injected into the peritoneal cavity

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of mice. Ab-richascites fluid was then collected, and the Abs were purified using

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proteinG-Sepharose (GE Healthcare) chromatography.

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2.6. Quantitative real-time PCR (Q-PCR)

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To obtain high-quality RNA, we prepared samples of fresh liver, intestine,

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supraneural body, and blood from each of three L.camtschaticum specimens. The

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extraction of leukocytes from the blood was performed as described previously (Pang

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et al., 2015). Total RNA was extracted from each L.camtschaticum tissue sample

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using TRIzol (Invitrogen, USA), and the RNA was treated with DNase I (TaKaRa,

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China). Reverse transcription was then performed as previously described (Pang et al.,

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2015). Q-PCR was conducted with the TaKaRa SYBR® PrimeScriptTM RT-PCR Kit

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according to the manufacturer’s protocol. Each reaction contained 1×SYBR Premix

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Ex Taq, 10 µM each primer, and 2 µL of cDNA (50 ng/µL) in a final volume of 25 µL.

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Amplification was performed in a TaKaRa PCR Thermal Cycler Dice Real Time

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System with the following parameters: initial denaturation at 95°C for 30 s to activate

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the DNA polymerase, followed by 40 cycles of 5 s at 95°C, 30 s at 55°C, and 30 s at

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72°C.

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5’-GCTGAAGGAGGGAGATGAGG-3’

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5’-AGTCGGGAAACAAGAGAAAACC-3’ (reverse). The GAPDH-specific primers

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were

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5’-GTCTTCTGCGTTGCCGTGT-3’

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dehydrogenase (GAPDH) was amplified as an internal control. Each sample was

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analyzed in triplicate using the Thermal Cycler Dice Real Time System analysis

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software (TaKaRa, China).

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2.7. Immunohistochemical staining

primers (forward)

5’-AACCAACTGCCTGGCTCCT-3’

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(forward)

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Glyceraldehyde3-phosphate

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Tissues were fixed overnight with 4% paraformaldehyde in PBS at 4°C and then

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embedded in paraffin. These paraffin-embedded specimens were sectioned at a

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thickness

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3-aminopropyltriethoxy silane (APES). The tissue sections were then de-waxed with

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xylene and dehydrated using a series of successively diluted alcohol. Endogenous

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peroxidase was inactivated by treating the sections with 0.3% hydrogen peroxide.

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Next, the sections were blocked with 10% normal donkey serum for 1 h at room

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temperature and incubated with the anti-rL-C1qDC-1 polyclonal Ab (1 µg/mL) for 3 h

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at room temperature, followed by the addition of HRP-conjugated goat anti-rabbit IgG

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(100-fold dilution) and rinsing with PBS. Normal rabbit IgG was used as a negative

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µm

and

collected

on

glass

slides

precoated

with

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ACCEPTED MANUSCRIPT control. Subsequently, the slides were stained with diaminobenzidine (DAB) and

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counterstained with hematoxylin. Following dehydration, the sections were passed

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through successively diluted concentrations of xylene for 15 min each and then

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mounted in neutral resin.

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2.8. Co-immunoprecipitation experiments and western blotting

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L.camtschaticum serum samples were incubated with 1 µg of an anti-VLRB mAb

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(or anti-IgG mAb) and the anti-rL-C1qDC-1 polyclonal Ab for 2 h at 4°C. Protein

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G-agarose was then added to each sample, followed by incubation at 4°C for 4 h and

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then centrifugation to collect the precipitated proteins. The samples were

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subsequently analyzed via 12% SDS-PAGE and transferred to NC membranes

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(Millipore).

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anti-rL-C1qDC-1 primary Abs, the proteins were detected with HRP-labeled goat

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anti-mouse or goat anti-rabbit secondary Ab and were revealed via ECL (Pierce,

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USA).

incubation

with

anti-VLRB

(or

anti-IgG)

and

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To explore the relationship between IgG and the L-C1qDC-1 proteins, we also used

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an NC membrane carrying IgG and L-C1qDC-1 proteins, which were previously

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separated via 15% SDS-PAGE. The NC membrane was blocked with 5% skim milk

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and then incubated with 40 µg/mL L-C1qDC-1 protein at 4°C overnight. Following

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incubation with anti-L-C1qDC-1 Ab, the NC membrane was washed and subjected to

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western blotting, as described above.

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2.9. Intracellular immunofluorescence staining

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ACCEPTED MANUSCRIPT HeLa and MCF-7 cells were treated with L.camtschaticum serum for 10 min at

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16°C. The cells were then washed twice with PBS, fixed and permeabilized for 10

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min using 0.1% Triton X-100, followed by blocking with normal 10% donkey serum

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for 2 h and incubation with 1 µg of anti-VLRB and anti-rL-C1qDC-1 Abs (200-fold

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dilution) at 4°C overnight. After overnight incubation, the cells were washed twice

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with PBS and then incubated with Alexa Fluor 488-labeled donkey anti-mouse IgG

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(400-fold) and Alexa 555-labeled donkey anti-mouse IgG (400-fold dilution).

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Following

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4’,6-diamidino-2-phenylindole (DAPI) (200-fold dilution). After two washes with

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PBS, the coverslips were mounted on glass slides along with drops of antifade

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solution. Immunofluorescence was then visualized and captured with a Zeiss LSM

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780 inverted microscope (Carl Zeiss, Inc.) and analyzed using Zeiss ZEN software. At

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the same time, we incubated MCF-7 cells with Alexa 488-conjugated VLRB and

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Alexa 555-conjugated L-C1qDC-1 protein for 2 h at 37°C in the dark, followed by

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three washes with PBS. Finally, 3D-SIM images of the cells were acquired on the

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DeltaVision OMX V3 imaging system (Applied Precision) using a 100×1.4 oil

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objective (Olympus UPlanSApo), solid-state multimode lasers (488 nm and 561 nm)

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and electron-multiplying charge-coupled device (CCD) cameras (Evolve 512×512,

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Photometrics).

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3. Results

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3.1. Cloning and amino acid sequence analysis of L.camtschaticum L-C1qDC-1

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cDNA

more

washes

with

PBS,

the

cells

were

stained

with

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ACCEPTED MANUSCRIPT We identified a fragment whose nucleotide sequence shared high homology with

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C1qDC homologs from other vertebrate species. Subsequent 3’-and 5’-RACE using

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primers designed based on this fragment resulted in the successful isolation of a

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full-length L-C1qDC-1 cDNA of 1,662 bp. This cDNA has a 711-bp ORF that

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encodes 236 amino acid residues, a 107-bp 5’-untranslated region (UTR) and an

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844-bp 3’-UTR with a polyadenylation signal (AATAAA). The amino acid sequence

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deduced from the L.camtschaticum L-C1qDC-1 cDNA demonstrated sequence

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similarity 41.9%-62.1% and identity 21.0%-42.4% from other vertebrates by the

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MatGAT software. Alignment revealed a strongly conserved C1q domain, which plays

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a significant role in binding a variety of ligands, at the C-terminus of the protein.

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L.camtschaticum L-C1qDC-1 contains a leading signal peptide whose splice site is

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between the seventeenth and eighteenth amino acids in the sequence, in addition to a

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collagen-like region and a globular C1q domain. In the L-C1qDC-1 protein, there is

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also an N-glycosylation site (Asn172) that contains the important modification function.

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BLASTx analysis showed that L-C1qDC-1 consistently shared 44% identity with the

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L.camtschaticum C1q-like protein, which demonstrated that L-C1qDC-1 is quite

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different from the C1q-like protein. Thus, L-C1qDC-1 is a novel C1q

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L.camtschaticum family member (Fig. 1).

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3.2. Phylogenetic analysis of L.camtschaticum L-C1qDC-1

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To investigate the evolutionary relationships between L.camtschaticum L-C1qDC-1

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and its counterparts, we constructed an NJ tree based on the amino acid sequences of

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C1qDC members. The tree suggested that all members of the C1q family can be 12

ACCEPTED MANUSCRIPT classified into two clusters. The first cluster is divided into three branches (C1qA,

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C1qB, C1qC), following lower to higher evolutionary relationships according to the

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evolutionary history. In the second cluster, the L.camtschaticum L-C1qDC-1 and the

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L.camtschaticum hagfish and sea urchin C1q-like sequences are clustered separately

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at the bottom of the evolutionary tree, indicating that they exhibit a unique position in

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the evolutionary history (Fig. 2).

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3.3. Expression and purification of the rL-C1qDC-1 protein

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To express recombinant L-C1qDC-1 in E. coli RosettaBlue (DE3), the mature

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protein-coding region, which encodes 236 amino acids of L-C1qDC-1, was generated

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via PCR and cloned between the EcoRI and HindIII sites of the pCold I vector.

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Amplification of the L-C1qDC-1 gene by PCR produced a single amplified 660-bp

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DNA fragment encoding a mature L-C1qDC-1 protein with a length of 236 amino

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acids. DNA sequencing confirmed that the cDNA sequence was identical to the

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reported sequence. Following induction with 0.1 mM IPTG, rL-C1qDC-1 was

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expressed as a His-tagged fusion protein in E. coli RosettaBlue (DE3). The purified

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rL-C1qDC-1 migrated as a single band in a 12% SDS-PAGE gel, with a molecular

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mass of approximately 26 kDa, consistent with the molecular mass predicted from the

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DNA sequence (Fig. 3).

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3.4. Titer and specificity analysis of Abs against the rL-C1qDC-1 protein

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Polyclonal Abs against the full ORF of the recombinant L-C1qDC-1 protein were

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generated through subcutaneous injection of male New Zealand white rabbits with the

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ACCEPTED MANUSCRIPT purified rL-C1qDC-1 protein. The titer of the obtained anti-L-C1qDC-1 serum was

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determined via ELISA at different dilutions (1,000-fold to 512,000-fold). The valence

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of the Abs secreted by the different clones was also assayed using ELISA. We chose

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clone MC-7, which has the highest valence of the sixteen clones, for our experiments.

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The specificity of the Abs was confirmed through western blot assays using the

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recombinant L-C1qDC-1 protein and serum from L.camtschaticum. In parallel, we

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used the entire gel separation range of 12% SDS-PAGE to examine the rL-C1qDC-1

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protein and total serum protein bands stained with Coomassie Brilliant Blue. The

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slight discrepancy between the predicted size and the actual size of the full-length

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L-C1qDC-1 protein, as determined by western blotting, may have been due to the fact

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that the pCold I vector is no more than 2kDa in size (Fig. 4).

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3.5.

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L.camtschaticum tissues

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expression

and

distribution

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of

L-C1qDC-1

in

different

The expression of L-C1qDC-1 mRNA was determined via real-time PCR using

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L-C1qDC-1-specific primers, as previously described. L-C1qDC-1 mRNA was highly

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expressed in the liver and intestine, whereas leukocytes and the supraneural body

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exhibited lower expression levels (Fig. 5A). Additionally, immunohistochemistry was

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performed with the anti-L-C1qDC-1 polyclonal Ab, and the results were identical to

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the Q-PCR results. Additionally, light microscopy revealed high L-C1qDC-1 protein

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expression in the liver and intestine of L.camtschaticum and lower expression in the

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supraneural body (Fig. 5B).

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3.6. Interaction of the VLRB and L-C1qDC-1 proteins The potential association between VLRB and L-C1qDC-1 was examined in

305

L.camtschaticum serum via co-immunoprecipitation. The results showed that the

306

anti-L-C1qDC-1 Ab could precipitate VLRB (a 35 kDa protein) from the serum,

307

similar to previously reported findings (Wu F, 2013). At the same time, the

308

anti-VLRB polyclonal Ab could precipitate L-C1qDC-1 (a 26 kDa protein) (Fig. 6A).

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Furthermore, we found that L-C1qDC-1 and VLRB were partially associated,

310

forming a complex in L.camtschaticum serum. When MCF-7 and HeLa cells were

311

incubated with L.camtschaticum serum, the complex of L-C1qDC-1 and VLRB was

312

deposited on the surface of the cells. In addition, fluorescent confocal analysis further

313

confirmed the deposition and interaction of VLRB and L-C1qDC-1 on the surface and

314

in the cytoplasm of target HeLa cells and MCF-7 cells (Fig. 6B). 3D-SIM images of

315

the cells also showed that the VLRB and L-C1qDC-1 proteins exhibited the same

316

orientation along the Z axis of observation (Fig. 6C).

317

4. Discussion

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All vertebrates, excluding agnathans, share a similar complement system. The

319

classical complement pathway has been suggested to have evolved from the lectin

320

pathway, although it adapted to become an Ig-recognizing system with the advent of

321

adaptive immunity during evolutionary history (Nayak et al., 2012). In

322

L.camtschaticum, an extant group of jawless vertebrates, the only component systems

323

appear to be the alternative and lectin pathways. Additionally, this group completely

15

ACCEPTED MANUSCRIPT 324

lacks the known major components of adaptive immunity. C1q is a target recognition protein of the classical complement pathway that serves

326

as a major link between innate and adaptive immunity. Recent findings have raised

327

the possibility that certain C1q-related proteins possess lectin activity and that

328

C1q-mediated complement activation occurs in even more primitive species (Yu et al.,

329

2008).

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In this study, we identified a novel L-C1qDC-1 gene in L.camtschaticum. Sequence

331

analysis illustrated that L-C1qDC-1 shared high homology with sequences from other

332

advanced vertebrates, whereas it only consistently shared 44% identity with the

333

L.camtschaticum C1q-like protein. Nevertheless, there are clear differences between

334

these sequences in terms of the structure of the tumor necrosis factor (TNF)

335

superfamily. However, these sequences all exhibit eight conserved domains that

336

constitute the hydrophobic core of C1q, which explains how C1q can bind to many

337

ligands. Further results of this study showed that the L-C1qDC-1 gene encodes 236

338

amino acid residues and contains a leading signal peptide, a collagen-like region, and

339

a gC1q domain that are extremely conserved. The L-C1qDC-1 protein also contains a

340

signal peptide with asplice site located at amino acids 17-18 and does not exhibit any

341

transmembrane structure; thus, it is likely a secretory protein. Additionally, there is an

342

N-glycosylation site in L-C1qDC-1. Due to the special status of L.camtschaticum,

343

further evaluation of the L-C1qDC-1 molecule will provide important information

344

regarding the function and evolution of the C1q family. To characterize this novel

345

molecule, we used real-time PCR to analyze its expression pattern. The mRNA

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ACCEPTED MANUSCRIPT expression of L-C1qDC-1 was highest in the liver and intestine and lower in the

347

supraneural body and leukocytes. Because the relative expression was very low in

348

other tissues of L.camtschaticum, we did not show this expression in detail.

349

Immunohistochemical staining assays more clearly demonstrated L-C1qDC-1

350

expression in the tissues, as mentioned above.

351

Jawless

fish,

and

especially

L.camtschaticum,

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exhibit

an

alternative

lymphocyte-based adaptive immune system based on somatically diversified

353

leucine-rich repeat (LRR)-based Ag receptors, referred to as VLRs. The

354

L.camtschaticum B-like lymphocyte lineage has been shown to express the

355

anticipatory receptor VLRB (Das et al., 2013). VLRBs are the counterparts of the Ig

356

that jawed vertebrates use for Ag recognition and complement activation.

357

L.camtschaticum lacks lymphatic organs, a spleen, and a thymus, and L-C1qDC-1

358

mRNA is distributed in the intestine, liver, leukocytes and supraneural body.

359

Interestingly, Cooper (Alder et al., 2008) reported VLRB expression in lymphocytes

360

from the blood, kidneys and typhlosoles. The somewhat similar distribution of the

361

L-C1qDC-1 protein and VLRB suggested that L-C1qDC-1 might also be involved in

362

the innate immune system, though whether it mediates complement activity is still

363

unknown. The blood circulation system is an important immune system for

364

transporting immune molecules and immune cells, and our results revealed interaction

365

of the L-C1qDC-1 protein with VLRB in L.camtschaticum serum. Moreover, analyses

366

using confocal microscopy and the DeltaVision OMX V3 imaging system revealed

367

the formation of complexes on the surface of target cells that might lead to

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ACCEPTED MANUSCRIPT 368

complement activation. Furthermore, we found that L-C1qDC-1 was primarily

369

located in the cytoplasm, and not in the nucleus. The complement system is a key component of the innate immune system and

371

plays an important role in the clearance of pathogens and apoptotic cells upon

372

its activation. It is well known that both IgG and IgM can activate the complement

373

system via the classical pathway through the binding of C1q to the Fc regions of these

374

Igs (Daha et al., 2011;Gadjeva et al., 2008). As previously stated, in humans and

375

mammals, C1q is the first subcomponent of the classical pathway of complement

376

activation and serves as a major link between classical pathway-driven innate

377

immunity and IgG-or IgM-mediated acquired immunity. Furthermore, in zebrafish,

378

C1qs can bind to heat-aggregated human IgG (Hu et al., 2010). Through

379

co-immunoprecipitation and western blotting experiments, we also found that there is

380

an interaction between the L-C1qDC-1 protein and IgG. It seems that L-C1qDC-1

381

exhibits a similar function to C1q in humans, playing an important role in immune

382

responses in L.camtschaticum (Supplementary Fig.1). We will further explore this

383

aspect in future research. Above all, these results indicated that L-C1qDC-1 might

384

primarily participate in VLRB-mediated and innate immune responses, which is key

385

to obtaining a detailed understanding of the origin and evolution of the complement

386

and immune systems.

387

5. Conclusions

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This report presents the characterization of a novel C1qDC protein (designated as

18

ACCEPTED MANUSCRIPT L-C1qDC-1) from L.camtschaticum. Expression pattern analysis showed that

390

L-C1qDC-1 participates in theimmune responses of L.camtschaticum. This work may

391

play an important role in achieving understanding of the universal functions of

392

C1qDC proteins in other species and suggests that these proteins could serve as

393

pattern recognition molecules in immunotherapy.

394

Acknowledgments

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This work was supported by the Chinese Major State Basic Research Development

395

Program (973 Program; Grant2013CB835304),

397

the Marine Public Welfare Project of State Oceanic Administration (No.201305016),

398

the Chinese National Natural Science Foundation (Grants 31170353 and 31202020), t

399

he Research Project of Liaoning Provincial Department of Education (No.

400

LJQ2014117) and the Science and Technology Project of Dalian (No. 2013E11SF056)

401

.

402

References

403 404 405 406 407 408 409 410 411 412 413 414 415 416

Alder, M.N., Herrin,B.R., Sadlonova, A., Stockard, C.R., Grizzle, W.E., Gartland, L.A., Gartland, G.L.,

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Boydston, J.A., Turnbough, C.L., Cooper, M.D., 2008. Antibody responses of variable lymphocyte receptors in the lamprey. Nat Immunol. 9, 319-327. Amemiya CT, Saha N.R., Zapata A., 2007. Evolution and development of immunological structures in the lamprey. Curr Opin Immunol. 19, 535-541. Bohlson, S.S., Fraser, D.A., Tenner, A.J., 2007. Complement proteins C1q and MBL are pattern recognition molecules that signal immediate and long-termprotective immune functions. Mol Immunol. 44, 33-43. Carland, T.M., Gerwick, L., 2010. The C1q domain containing proteins: Where do they come from and what do they do? Dev Comp Immunol. 34, 785-790. Daha, N.A., Banda, N.K., Roos, A., Beurskens, F.J., Bakker, J.M., Daha, M.R., Trouw, L.A., 2011. Complement activation by (auto-) antibodies. Mol Immunol. 48, 1656-1665. Das, S., Hirano, M., Aghaallaei, N., Bajoghli, B., Boehm, T., Cooper, M.D., 2013. Organization of lamprey variable lymphocyte receptor C locus repertoire development. Proc Natl Acad Sci USA. 110,

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Kishore, U., 2007.C1q and its growing family. Immunobiology. 212, 253-266.

Han, B.W., Herrin, B.R., Cooper M.D., Wilson I.A., 2008. Antigen recognition by variable lymphocyte receptors. Science. 321, 1834-1837.

Hu, Y.L., Pan, X.M., Xiang, L.X., Shao, J.Z., 2010. Characterization of C1q in teleosts: insight into the molecular and functional evolution of C1q family and classical pathway. J Biol Chem. 5, 28777-28786.

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Kishore, U., Ghai, R., Greenhough, T.J., Shrive, A.K., Bonifati, D.M., Gadjeva, M.G., Waters, P., Kojouharova, M.S., Chakraborty, T., Agrawal, A., 2004. Structural and functional anatomy of the globular domain of complement protein C1q. Immunol Lett. 95, 113-128.

Kishore, U., Reid, K.B., 2000. C1q: structure, function, and receptors. Immunopharmacology. 49,

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Matsushita, M., Matsushita, A., Endo, Y., Nakata, M., Kojima, N., Mizuochi, T., Fujita, T., 2004. Origin of the classical complement pathway: Lamprey orthologue of mammalian C1q acts as a lectin. Proc Natl Acad Sci U S A. 101, 10127-10131.

Nayak, A., Ferluga, J., Tsolaki, A.G., Kishore, U., 2010. The non-classical functions of the classical complemet pathway recognition subcomponent C1q. Immunol Lett. 131, 139-150. Nayak, A., Pednekar, L., Reid K.B., Kishore, U., 2012. Complement and non-complement activating

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functions of C1q: a prototypical innate immune molecule. Innate Immun. 18, 350-363. Osório, J., Rétaux, S., 2008. The lamprey in evolutionary studies. Dev Genes Evol. 218, 221-235. Pang, Y., Liu, S., Zheng, Z., Liu, X., Li, Q., 2015. Identification and characterization of the lamprey IRF gene. Immnol Lett. 164, 55-64.

Pang, Y., Xiao, R., Liu, X., Li, Q., 2012. Identification and characterization of the lamprey

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high-mobility group box 1 gene. PloS One. 7, e35755. Tahtouh, M., Crog, F., Vizioli, J., Sautiere, P.E., Van, C.C., Salzet, M., Daha, M.R., Pestel, J., Lefebvre, C., 2009. Evidence for a novel chemotatic C1q domain-containing factor in the leech nerve cord. Mol

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Wang, L., Wang, L., Zhang, H., Zhou, L., Siva, V.S., Song, L., 2012. A C1q domain conataining protein from scallop Chlamys farreri serving as pattern recognition receptor with heat-aggregated IgG binding activity. PloS One. 7, e43289. Wu, F., C, L., Liu, X., Wang, H., Su, P., Han, Y., Feng, B., Qiao, X., Zhao, J., Ma, N., Liu, H., Zheng, Z., Li, Q., 2013. Lamprey variable lymphocyte receptors mediate complement-dependent cytotoxicity. J Immunol. 190, 922-930. Yu, Y., Huang, H., Wang, Y., Yu, Y., Yuan, S., Huang, S., Pan, M., Feng, K., Xu, A., 2008. A novel C1q family member of amphioxus was revealed to have a partial function of vertebrate C1q molecule. J Immunol. 181, 7024-7032.

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ACCEPTED MANUSCRIPT Fig. 1 Sequence alignment of L.camtschaticum L-C1qDC-1 and C1q-like

461

proteins.

462

A. L.camtschaticum L-C1qDC-1 and C1q-like molecules. BLASTx analysis of the

463

L-C1qDC-1 and C1q-like sequences showed that they consistently shared only 44%

464

identity.

465

http://smart.embl-heidelberg.de/. Signal peptides are highlighted in red; the

466

collagen-like region, in yellow; and the C1q domain, in green. The blue boxes mark

467

the eight conserved amino acid residues, and the red box represents the

468

N-glycosylation site.

469

B. Stick models of the functional domains in the L-C1qDC-1 gene. The three

470

loop-like regions indicate the omitted amino acids.

471

Fig. 2 Phylogenetic tree for L-C1qDC-1 based on the NJ method.

472

An NJ tree was constructed using the amino acid sequences of C1qDC proteins. The

473

numbers at the nodes indicate the bootstrap confidence values derived from 1,000

474

replications. The bar (0.1) indicates genetic distance. The accession numbers of the

475

amino acid sequences extracted from the NCBI protein database are as follows:

476

Human C1qA (P02745.2); Mouse C1qA (P98086.2); Rat C1qA (P31720.2); Bovine

477

C1qA (Q5E9E3.1); Dog C1qA (XP535367.1); Horse C1qA (XP001504311.1);

478

Opossum C1qA (XP001376435.1); Pig C1qA (Q69DL0.1); Rhesus C1qA

479

(XP001101837.1); Chicken C1qA (XP417654.2); Zebrafish C1qA (ACN62221.1);

480

Human C1qB (P02746.3); Mouse C1qB (P14106.2); Rat C1qB (P31721.2); Bovine

functional

domains

of

L-C1qDC-1

are

according

to

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ACCEPTED MANUSCRIPT C1qB (Q2KIV9.1); Dog C1qB (XP544507.2); Horse C1qB (XP001501545.1);

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Rhesus C1qB (XP001110783.1); Chicken C1qB (XP425756.2); Human C1qC

483

(P02747.3); Mouse C1qC (Q02105.2); Rat C1qC (P31722.2); Dog C1qC

484

(XP544508.2); Horse C1qC (XP001504308.1); Rhesus C1qC (XP001102196.1);

485

Chicken C1qC (XP417653.1); Zebrafish C1qC (ACN62223.1); Shark C1q-like

486

(AFK11213.1); Lamprey C1q-like (BAD22833.1); Hagfish C1q (BAM76761.1);

487

Urchin C1q-like (XP782700.1).

488

Fig. 3 SDS-PAGE analysis of the rL-C1qDC-1 protein expressed in E. coli

489

RosettaBlue.

490

A. SDS-PAGE analysis of the rL-C1qDC-1 protein expressed in E. coli RosettaBlue.

491

M, low-molecular-weight protein marker; Lane 1, induced expression of

492

RosettaBlue/pCold I; Lane 2, non-induced expression of RosettaBlue/pCold

493

I-L-C1qDC-1; Lane 3, induced expression of RosettaBlue/pCold I-L-C1qDC-1; Lane

494

4, supernatant; Lane 5, inclusion body.

495

B. Purified rL-C1qDC-1 protein. M, low-molecular-weight protein marker; Lane 1,

496

supernatant fluid from the sonicated bacterial cells; Lane 2, flow-through sample;

497

Lane 3, washing sample; Lane 4, the purified recombinant protein.

498

Fig. 4 Specificity analysis of Abs against the rL-C1qDC-1 protein.

499

Western blot analysis of the specificity of rabbit anti-L-C1qDC-1 polyclonal Abs and

500

mouse anti-L-C1qDC-1 mAbs. A. SDS-PAGE analysis of rL-C1q-DC-1 protein and

501

serum from L.camtschaticum. B. Western blotting showing the specificity of the

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ACCEPTED MANUSCRIPT anti-rL-C1qDC-1polyclonal Abs. C. Western blotting showing the specificity of the

503

anti-rL-C1qDC-1mAb. M, pre-stained protein ladder; L-C1qDC-1, the purified

504

rL-C1qDC-1 protein; Serum, serum from L.camtschaticum.

505

Fig. 5 Q-PCR analysis and immunohistochemical staining of the L-C1qDC-1

506

protein in L.camtschaticum tissues.

507

A. Q-PCR analysis of the L-C1qDC-1 gene in L.camtschaticum, characterizing

508

L-C1qDC-1 mRNA expression in adult tissues. The mRNA levels of L-C1qDC-1 are

509

expressed as a ratio in relation to GAPDH mRNA levels, n=3.

510

B. a. Localization and distribution of the L-C1qDC-1 protein in L.camtschaticum

511

tissues, as observed via immunohistochemical staining. The tissue sections were

512

incubated with the anti-L-C1qDC-1 polyclonal Ab at 8 µg/mL. Normal rabbit IgG

513

served as a control. The tissues included the supraneural body, liver, and intestine.

514

Positive cells are indicated by red arrows. Scale bars: 50 µm, magnification: 40×, n=3.

515

b. Histogram showing statistics of the above results.

516

Fig.6

517

co-immunoprecipitation and laser scanning confocal microscopy analysis.

518

A. Co-immunoprecipitation of L-C1qDC-1 and VLRB from L.camtschaticum

519

serum.The anti-L-C1qDC-1 Ab precipitated the VLRB protein (35 kDa, Fig. a), and

520

the anti-VLRB mAb precipitated the L-C1qDC-1 protein (25 kDa, Fig. b).

521

S:supernatant; P:precipitation.

522

B. Colocalization of L-C1qDC-1 and VLRB, asdetermined via laser scanning

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of

L-C1qDC-1

and

VLRB,

as

determined

via

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23

ACCEPTED MANUSCRIPT confocal microscopy analysis. MCF-7 and HeLa cells incubated with Abs. The

524

primary Abs consisted of mouse anti-VLRB mAbs and mouse anti-L-C1qDC-1 mAbs,

525

and the secondary Abs consisted of Alexa 488-labeled donkey anti-mouse IgG and

526

Alexa 555-labeled donkey anti-mouse IgG. The nuclei were stained with DAPI. Scale

527

bar, 5 µm.

528

C. Colocalization of L-C1qDC-1 and VLRB, as determined using the DeltaVision

529

OMX V3 imaging system. MCF-7 cells incubated with Alexa 488-conjugated VLRB

530

protein and Alexa 555-conjugated L-C1qDC protein. Scale bar, 5 µm.

531

Supplementary Fig. 1. Interaction of L-C1qDC-1 and IgG, as determined via

532

western blotting and co-immunoprecipitation.

533

A. Co-immunoprecipitation of L-C1qDC-1 and IgG. The anti-IgG mAbs precipitated

534

the L-C1qDC-1 protein (25 kDa) of L.camtschaticum serum, and anti-L-C1qDC-1 Ab

535

precipitated the L-C1qDC-1 protein as a negative control.

536

B. Interaction of L-C1qDC-1 and IgG, as determined via western blotting. The

537

L-C1qDC-1 and IgG proteins were separated by SDS-PAGE and then transferred to

538

an NC membrane and incubated with L-C1q-DC-1 (40 µg/mL). Bound L-C1qDC-1

539

was detected with an HRP-conjugated anti-L-C1qDC-1 Ab.

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Sequences 5'-GAGCCGCAGTACAACACAGTTCAAG-3' 5'-CAGGAAGCCGAGGTGAACCAGGATT-3' 5'-GATCTTGGCAGTGAAGGCTGACTTTGGC-3'

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5'-AAGTCTATTTTGTCTGCTTTCGGGCCGG-3' 5'-TTCAGCGGAAAGCCACGAGGAC-3' 5'-TCTATTTTGTCTGCTTTCGGGC-3‘

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Primers. Name 3'RACE-F1 3'RACE-F2 5'RACE-R1 5'RACE-R2 L-C1qDC-1 F L-C1qDC-1 R

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Tab1. Specific primer sequences

ACCEPTED MANUSCRIPT

Fig. 1

B

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A

5’UTR

SP

Collagen

C1q

3’UTR

ACCEPTED MANUSCRIPT

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Fig. 2

ACCEPTED MANUSCRIPT

A. M

1

2

3

4

5

KDa 97.2

97.2

66.4

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66.4

44.3

20.1

14.4

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44.3

29.0

M

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KDa

B.

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Fig. 3

29.0

20.1

14.4

1

2

3

4

ACCEPTED MANUSCRIPT

Fig.4

M L-C1q-DC-1

Anti-rL-C1q-DC-pAb Serum

L-C1q-DC-1 Serum A

SC M AN U EP AC C

L-C1q-DC-1

B

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35KDa 25KDa

Anti-rL-C1q-DC-mAb

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SDS-PAGE

Serum C

ACCEPTED MANUSCRIPT

1.8 1.6

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Relative expressior level of L-C1q-DC-1

2

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Fig.5A

M AN U AC C

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L-C1qDC-1

GAPDH

1.4 1.2 1

0.8 0.6 0.4 0.2 0

Intestine

Leukocyte Supraneural body

Liver

ACCEPTED MANUSCRIPT

Liver

Supraneural body

SC M AN U TE D EP AC C

Anti-L-C1qDC-1-antibody

b. Relative expression of L-C1qDC-1 protein

Intestine

Normal-rabbit-IgG

a.

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Fig. 5B.

450 400 350 300 250 200 150 100 50 0

Intestine

Liver

Supraneural body

ACCEPTED MANUSCRIPT

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Fig. 6A a.

Anti-L-C1qDC-1 S P

SC

Sera

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VLRB

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L-C1qDC-1

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Sera

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b.

S

40KDa 35KDa

Anti-VLRB P 35KDa 25KDa

ACCEPTED MANUSCRIPT

VLRB

L-C1qDC-1

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MCF-7

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Hela

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Nuclear

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Fig.6B Merge

ACCEPTED MANUSCRIPT

L-C1qDC-1 VLRB

L-C1qDC-1 VLRB

L-C1qDC-1 VLRB

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L-C1qDC-1 VLRB

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Fig. 6C

Z-axis

ACCEPTED MANUSCRIPT

Anti-IgG P

Anti-L-C1qDC-1 P

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A.

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Supplymentary Fig.1

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L-C1qDC-1

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25KDa

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L-C1qDC-1 IgG L-C1qDC-1

B.

ACCEPTED MANUSCRIPT

Highlights 1. This report describes the identification and characterization of a novel C1qDC protein (designated as L-C1qDC-1) from Lethenteron camtschaticum. 2. This report represents that L-C1qDC-1 participated in VLRB-mediated and innate

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immune responses, and it is important to understand the origin and evolution of the complement and immune systems. It may play an important role of

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understanding the universal functions of C1qDC proteins in other species.