Limited expression in human tissues

Biochem.J. (1991) 276,369-379 (Printedin GreatBritain)

Primary structure of the human laminin A chain

Limited expression in human tissues

Marja NISSINEN,* ReettaVUOLTEENAHO,* RaymondBOOT-HANDFORD,t Pekka KALLUNKI* and KarlTRYGGVASON*1

*Biocenter and Department of Biochemistry, University ofOulu, SF-90570 Oulu, Finland, and

tDepartmentofBiochemistry andMolecular Biology, UniversityofManchester, Oxford Road, Manchester M13 3PT, U.K.

cDNA clones for the human laminin A chain were isolated from libraries prepared from human gestational choriocarcinoma cell line (JAR) RNA. Theycover approx. 8kb from the 5'-end of the9.5kb mRNA coding for this protein. Our clones contain 94 nucleotide residues for the 5'-end untranslated region and 7885 nucleotide residues of coding sequence. The complete human laminin A chain contains a 17-amino acid-residuesignal peptide and a 3058- residue Achainproper.The human laminin A chain hasadistinct domainstructurewithnumerousinternal cysteine-rich repeats. The large globular domain G has five repeats, which have several conserved glycine and cysteine residues.

Furthermore the Achain contains 20 internal cysteine-rich repeats present in tandemarrays in threeseparate clusters (domains IIIa, IIIb and V). Domain I+IIhasapredicted continuousa-helicalstructurecharacterized by heptadrepeats

andthree domains (IVa, lVb and VI)arepredictedtocontainanumberof f-sheets and coiled-coilstructures.Northern-blot analysiswas usedto study the laminin Achain expression in the JAR cell line, full-term placenta and newborn-human tissues (kidney, spleen, lung, heart muscle, psoas muscle and diaphragm muscle). The expression was detectable in newborn-humankidney and JAR cell lineonly. The overall amino acidsequenceidentity between human and mouseis 76%. The human chain has onlyoneArg-Gly-Asp (RGD)sequence,which islocated in the longarmwithindomain G, whereas thesingle RGDsequenceinthemousechain is located in the shortarmin domain IlIb. The degreeof identity between the human laminin A chainsequenceand thesequenceavailable for merosin[Ehrig, Leivo, Argraves, Ruoslahti

&Engvall (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3264-3268] is about 41 % and when conservative substitutions are

included thedegree of similarity is 54 %.

INTRODUCTION

Laminin isaspecificcomponentof basement membranes that has been showntomediateanumber ofbiological functions such

ascelladhesion, growth, migration and differentiation [1,2]. It has been shown to bind to cells through membrane receptors [3-5]. The laminin molecule isaverycomplex protein composed of three chains that formacruciformstructurewith three short

arms and onelong arm [2,6]. The molecule hasone heavy (A) chain(Mrapprox.400000) andtwolight (B) chains(Mrapprox.

200000). To date,tworelatedheavy chains have been described, the^socalledAchain[1,2]and merosin[7,8].Furthermore,there

arethreetypesoflight chains,B1,B2[1,2]and s-laminin[9].The complete primarystructureof themouseAchain[10]andapart of thehuman A chain[11]and merosin[8]have been determined.

Also, thesequences ofthe Bi and B2 chains fromman[12,13],

mouse [14,15] and Drosophila [16,17] and the s-laminin chain from rat[9] have been reported. All the chains containglobular domains, rod-like domains withcysteine-rich repeats andanaz-

helical domain thatparticipatesin thetriplecoiled-coilstructure forming the longarmof the molecule.Additionally, theAchain hasalargeC-terminalglobule, termed domain G[2,10,18]. The existence ofmultiplelaminincomponent chains enablesavariety ofmolecular compositions whose nature, distribution and bio- logical significanceareonly beginningtobecomeunravelled. For example, the A chain has been shown to be expressed to a

considerably smaller extentin adultmouse tissues thanthe Bl and B2 chains[19], and Schwanncells maysynthesize alaminin

molecule notcontaininganAchain [20]. Merosin ispresent in human placenta and Schwann cells [7,21]. Merosin isnotfound in themousefetus but appearspostnatally in muscle andnerve

[7,21]. The Bl and B2 chainsareexpressed inmosttissues that contain basement membranes, but their ratio can vary con-

siderably [22-24]. Furthermore, the s-laminin chain is primarily foundinsynaptic basement membranes but is also found in the kidney [9]. A distinct 300000-M, laminin heavy chain in heart muscle has also been described [25].

Proteolytic-digest fragments, synthetic peptides and domain- specific antibodies have been used to dissect the biologically activeregions of themouseEngelbreth-Holm-Schwarm-tumour laminin molecule and itscomponentchains. These studies have revealed twocell-binding domains, one inthe central region of thecross(P1 fragment) and anotheroneinthelongarm[2,26-29].

It has been suggested that a Tyr-Ile-Gly-Ser-Arg (YIGSR)

sequence indomainIII in theBi chain[29]oranArg-Gly-Asp (RGD)-sequence-containing region in domain IIlb of the A chain[27]isresponsiblefor the cellbindingof the P1 fragment.

Accordingto Deutzmannetal. [30],amajorcell-binding site is also located in end of thelongarmof laminin.Also,fragments from thelongarmhave been showntostimulate neuraloutgrowth [31,33], andaheparin-bindingdomain [34]has beenassignedto the globular domain of the A chain. Furthermore laminin fragmentsfrom the shortarmhave been showntostimulate cell growth [35],and the A chainmayplayarole in thepolarization ofepithelialcells [36].

Comparison ofsequences of similar proteins from different

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369

t To whomcorrespondence shouldbeaddressed.

The nucleotidesequencedatareportedwillappearin theEMBL,GenBank and DDBJ NucleotideSequenceDatabases under the accession number X58531.

speciesisimportanttofindconserved,andtherefore, presumably biologically important, regions. In an attempt toelucidate the structure, function and expression of laminin, we have isolated cDNA clonesfor thehumanAchain anddetermined its complete structure. The results showed that the human chain has con- siderable sequence identity with the mouse chain, and also considerable similaritytothehumanmerosin chain. Expression wasshown to be negligible in or absent from a number of human newborn tissuesexcept kidney.

METHODS

Isolation andidentification of mouse lamininAchain cDNA clone

Inorder to obtain aprobe to screen for the human Achainwe first isolated a mouse laminin A chain cDNA clone. A Agtl 1 cDNA library containing inserts derived from mouse parietal endoderm mRNA (obtained from Dr. Markku Kurkinen, U.M.D.J.N.-Robert Woods-Johnson Medical School, Piscataway, NJ, U.S.A.) was immune-screened with a rabbit polyclonal antiserum to Engelbreth-Holm-Schwarm-tumour laminin(provided byDr. P.Brenchley,UniversityofManchester, Manchester, U.K.). Positive clones were screenedto purity and the cDNA inserts were characterized initially on the basis of restriction mapping and the size of mRNA recognized on hybridization to Northern blots oftotal RNA obtained from induced F9 embryonal teratocarcinoma cells [37]. A 2.5 kb cDNA EcoRI insert (AlamAl) that hybridized to an inducible 10 kb mRNAfrom F9cellswas subcloned into theM13mpl8vector, and sequencing revealed its origin to be an internal EcoRI fragment (nucleotide residues 6641-9065) of the full-length mouse laminin Achain [10].

Preparation of cDNAlibrariesfrom human JAR cells

cDNA libraries were made by using polyadenylated RNA fromahumangestationalchoriocarcinoma(JAR)cellline (kindly providedbyDr. G. P.Frenette,University ofMichigan Medical School,AnnArbor,MI,U.S.A.) that secretes the laminin A chain [38,39] and whose mRNA hybridized with the mouse cDNA clone AlamA- 1 (results not shown). The polyadenylated RNA was isolated directly from cultured JAR cells with a slight modification of the Fast Track RNA Isolation Kit method (Invitrogen, San Diego, CA, U.S.A.). Briefly, the cellswerelysed with a buffer containing proteinase K and incubated with oligo(dT)-cellulose. The non-polyadenylated RNA, DNA and proteins were washed off in buffer containing LiCl and the polyadenylated RNAwaselutedinlow-salt buffer.Anoligo(dT) primer cDNA library was madefrom the JAR polyadenylated RNA by using cDNA synthesis and Agtl1 cloning system kits (Amersham International, Amersham, Bucks., U.K.). Briefly, the cDNA wasmethylated, ligated toEcoRI-linkers, separated from the linkers after EcoRIdigestion on a Sephadex column, ligated into the Agtl1 vector and packaged by using Gigapack extracts (Stratagene, La Jolla, CA, U.S.A.). A first primer extension library was constructed with the JAR cell polyadenylated RNA.The cDNAwaspreparedwith the cDNA synthesis kit (Amersham International) using a synthetic oligonucleotide primerCl witha sequence (nucleotide residues 5540-5465) from the 5'-end of theA-1 clone, isolated from the first cDNAlibrary.In contrastwith theoligo(dT)-primedlibrary, the cDNA was purified by using cDNA Spun Columns (Pharmacia, Uppsala,Sweden)and cloned into theAgtl1 vector

by using an EcoRI adaptor system (Promega, Madison, WI, U.S.A.) that doesnotrequiremethylation and EcoRI digestion of the cDNA before ligation into the Agtll vector. A second

primer extension cDNA library was then similarly prepared by using a synthetic primer C2 with a sequence (nucleotide residues 3422-3449) from the 5'-end region of the longest clone, C1-23, isolatedfrom the first extension cDNA library. The cDNA was purified and cloned into a AgtlO vector (Promega) by using EcoRI/NotI adaptors (Pharmacia).

Isolation and characterization of human lamininAchain cDNA clones

The oligo(dT)-primed JAR cell line cDNA library was screened with the 32P-labelled mouse cDNA clone MamA-1 by using standardprotocols [40]. The first primer extension library made with the Cl primer was screened with the longest 32P-labelled clone (A-1) isolated from the JAR cell line cDNA library. The second primer extension library generated with the C2 primer wassimilarlyscreened with thelongest 32P-labelled cloneC1-23 isolated fromthe first primerextension library. Purified cDNA clones were isolated, subcloned into pUC18/19 plasmids and M13mpl8/19 vectors with standard methods [40] and sequenced from both strandswith thedideoxynucleotide chain-termination procedure [41].

Northern-blot analysis

Humantissuesamples wereobtainedat autopsyfroma6-day- old infant within 6 hafter death (provided by Dr. Ritva Herva, University Hospital of Oulu, Oulu, Finland) and immediately frozen in liquid N2. Total RNA was isolated [42], and about a 20,ugsamplewaselectrophoresedin aformaldehyde-containing gel,blottedon tonitrocellulose filtersandhybridizedwith nick- translated cDNA inserts [40]. As probe for human laminin A chain expression the 2.5kb A-1 clone (nucleotide residues 5320-7835; see Fig. 2) was used. For analysis of Bi chain expressionanXbaI-BamHI(2.3kb)fragment of fromthe5'-end of thecDNA sequence(nucleotide residues ^-180-2157)[12] was used,andforthe B2chainthe 2.4 kb HL-205 cDNAcontaining nucleotide residues 2485-4954 [13] was used.

RESULTS

Isolationandcharacterization of cDNA clones

Initialscreening ofseveral commercial human cDNA libraries from placenta, fibroblasts, endothelium, HT-1080 cells and keratinocytes (Clontech Laboratories, Palo Alto, CA, U.S.A.) thatwereshown tocontaincDNA clonescodingfor laminin B1 and B2 chains ([12,13]; M.Nissinen, unpublished work) did notyield any positive signals for laminin A chain clones when screened with lamininpolyclonalantibodies orwiththe approx.

2.5kb mouseAlamAl cDNA clone. However, hybridization of themouselaminin A chain cDNA clone(AlamAl)to anapprox.

9.5kb mRNAfromthe humangestational choriocarcinomacell line JAR(results notshown)indicated significant expressionof the laminin A chain gene. This cell has been showntosynthesize significant amounts of laminin containing the A chain [38,39].

Thereforeanoligo(dT)-primed JAR cell line cDNAlibrarywas screened with themousecDNAclone, AlamAl.Thisresulted in the isolationofone2.5kbclone, A-1 (Fig. 1),whichhybridized with theexpected9.5 kb-size mRNAfromthe JAR cells(seeFig.

3). Initial nucleotidesequencingfromboth ends of the clone and comparisonwith themouse[10]and3'-endhuman sequence[11]

revealed that the A-1 cDNA coded for the human laminin A chain. The A-1 cDNA cloneoverlapped 698bpwith the 5'-end ofa previously reported [11] cDNA clone. The fact that the A-I cDNA clone didnotcontainthe3'-end withapoly(A) tail, although the library was made with an oligo(dT) primer, is probablyduetoinefficientmethylationof theinternalEcoRI site

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Fig. 1.cDNA clonescodingthehumanlaminin Achain,and theirpartialrestrictionmapand the domainstructureof theprotein

ATG indicates thetranslation initiationsignal,and TGA the 3'-end translationstopcodon[11]. Restriction-enzymesitesEcoRI(E),HindIll(H) and PstI(P)areshown. The domainsaremarkedaccordingtoSasakietal. [10].Five internalrepeatsin domainGareindicatedbyhatched boxes.

Domains Illa,Illband Vcomposedofcysteine-richinternalrepeatsareshownby stippledboxes.

in the cDNAsequence (Fig. 1)sothat the 3'-end fragment was

lost during the cloning of the original cDNA into the Agtl library vector.

The first primer extension cDNA library was then made to obtain more 5'-end clones by using an oligonucleotide primer containing sequences from the 5'-end region of the A-1 cDNA clone. This librarywas screened with theA-1 cDNA, and four

newcDNAclones, C1-24, C1-20, Cl-29 and C1-23,wereisolated and characterized (Fig. 1). The C 1-23 cDNA clone was the longestone,containinganinsert of 2.2 kb. A 5'-endsequenceof C1-23 was similarlyused toprepare the second specific cDNA libraryasdescribed in the Methodssection, and screening ofthis library with the C1-23 cDNA resulted in the isolation of cDNA clones C2-8, C2-10 and C2-12 (Fig. 1). The C2-12 cDNA was

3.6kb, and it contained sequence coding for the 5'-end untranslated region of the mRNA, the signal peptide and theN- terminal end of the mature laminin A polypeptide chain.

Altogether, the clones isolated and characterized in this study spanned 8.0 kb fromthe 5'-end of the 9.5 kbmRNA, and thus, together with the previously reported 2.5 kb 3'-endsequence[11], they provide the coding sequence andprimary structure ofthe entire human lamininAchain.

Nucleotide and amino acidsequences

Thecomplete nucleotidesequenceof the cDNA clones studied here is shownin Fig. 2. together with the deduced amino acid

sequence. The previously reported [11] 680-residue C-terminal aminoacid sequence(residues 2379-3058; Fig. 2) isalso shown toprovide here the completesequenceof theAchain. Differences in the amino acid sequence between the human and mouse A chains ^are also indicated. The sequence of the overlapping cDNA clones characterized in thisstudycontains 95 nucleotide residues coding for the 5'-end untranslated region and 7885 nucleotide residuesofopenreadingframe. There isaputative 17- residue signal peptide composed of the translation initiator methionine residue followed by a typical hydrophobic leucine- rich sequence that contains two cysteine residues. The signal peptideof themousechain is 24 residues inlengthand contains

nocysteineresidues. The N-terminal end of thematuremouseA chain, determined byamino acid sequencing [43], startswith a

glutamine residue whereas the equivalent residue in the human

chain isarginine.

ThematurehumanAchain mRNA encodes 3058amino acid residues, two less than themouse counterpart. The A chain has

adistinct domainstructurewithnumerous internalcysteine-rich repeats,ashas been described for the^mouseAchain[10]. There isalarge domain G of 950 residues (residues 2109-3058)atthe C-terminal end(Fig. 1). Thealignedamino acidsequenceof the human and mouse A chains (Fig. 2) shows that the human domain G has one extra residue (serine, residue 2523), but is missing two other residues found in the mouse (proline and glutamine atpositions 2681 and 2682). The degree ofsequence

identity in domain G between man and mouse is 76%. The human domainI+II (residues 1539-2108) has one residue less than themousecounterpart. Histidine-1735 in the humanchain doesnothaveacounterpartinthemousechain, whereas valine- 1658 and glutamic acid-1775 in the mouse chain do not have matches in the human chain. The degree ofsequence identity between this domainin man and^mouse is 63%. The A chain contains 20 internal cysteine-rich repeats present in a tandem

arrayinthreeseparatecluster, domains IlIa (residues 1345-1538), IIb (residues 692-1142) and V (residues 253-495). The degrees ofamino acidsequenceidentityof theIlla,IlIbandVdomains between manandmouse are 73%, 81% and 79% respectively.

The globular domains IVa(residues 1143-1344), IVb (residues 496-691) and VI (residues 1-252) have degrees of sequence

identity between man and mouse of 79%, 84% and 88% respectively.

The overall degree of amino acid sequence identity between the human andmousechainswascalculatedtobe 76%.All 164 cysteineresidues with theexceptionofone(2561) ^areconserved between thetwospecies. Also,23outof 30potentialattachment sites forasparagine-linked oligosaccharides inthe human chain

areconserved in themouse. Interestingly, the humanchain has only^oneRGDsequence,which is located in thelongarmwithin domain G (residues 2517-2519), whereas the single RGD

sequence in the mouse chain [10] is located in the short arm

withindomain IlIb at locationresidues 1123-1125.

Expressionof lamininA,Bi andB2 chainsinhuman tissues The expression of the laminin A chain gene was compared with that of BI and B2 chain genes in several human tissues.

Total RNA isolated from newborn-humankidney, liver, spleen, lung, heart muscle, diaphragm muscle, psoas muscle and full- termplacentatissuestogetherwith RNAfrom the JAR cellswere

size-fractionated by agarose-gel electrophoresis, blotted on to nitrocellulose andhybridizedwithnick-translated human laminin A-chain, BI-chain and B2-chain probes. The results showed a C2-12

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