Identification and recombinant production of human laminin α4 subunit splice variants

https://doi.org/10.1016/S0006-291X(02)02642-6Get rights and content

Abstract

Laminins, the major basement membrane glycoproteins, are composed of three subunits. We identified a splice variant of the human laminin α4 subunit transcript containing 21 extra nucleotides. A heptapeptide sequence, MDCPTIS, was inserted close to the two cysteine residues possibly involved in the intersubunit disulfide bonds. Both the authentic α4 subunit (α4A) and the variant with the heptapeptide insertion (α4B) were readily secreted as laminin-8 trimers (α4Aβ1γ1 or α4Bβ1γ1) upon cotransfection with expression vectors for the β1 and γ1 subunits. The purified recombinant laminin-8 containing the α4B subunit was more potent in promoting cell spreading than that containing α4A, raising the possibility that the alternative splicing of the α4 subunit transcript regulates the cell-adhesive activity of laminin-8. Since both α4A and α4B transcripts were detected by RT-PCR in several human cell lines, these two isoforms of laminin-8 with differing cell-adhesive activities are present in the basement membranes of human tissues.

Section snippets

Materials and methods

Cells and cell culture. The following cell lines were obtained from American Type Culture Collection (Manassas, VA): SK-N-SH and NB-1 (neuroblastoma); T98G, U251MG, U251SP, and AJ (glioma); HepG2 (hepatoblastoma); JEG3 (choriocarcinoma); A549 (lung carcinoma); 293 (embryonic kidney epithelial cells); and HT1080 (fibrosarcoma). All cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma) supplemented with 10% fetal bovine serum (FBS) under an atmosphere of 95% air/5% CO2 and

Identification of a splice variant of human laminin α4 cDNA

A fragment of laminin α4 subunit cDNA with 21 extra bases was identified by sequencing the RT-PCR products amplified from the T98G glioma cells (Fig. 1A). This 21-bp insertion results in an in-frame insertion of seven amino acid residues, MDCPTIS, between Gly265 and Cys266 (amino acids are numbered according to [5]). Interestingly, a cysteine residue is present within the seven amino acid insert which is located very close to two other cysteines, Cys266 and Cys269. Since these cysteine residues

Cited by (20)

  • Laminin isoform profiles in salivary glands in Sjögren's syndrome

    2011, Advances in Clinical Chemistry
    Citation Excerpt :

    Cytokines and other molecules regulating LM transcription bind to enhancer or silencer elements found in promoters of LM genes, in the 5′ untranslated region, or inside introns as is the situation with murine LAMC1[57–60]. Additional dimension to LM regulation comes from alternative splicing, which has been demonstrated in almost all LM chains resulting in multiple isoforms of these chains [61–64]. Further, promoter methylation of LAMA3, LAMB3 and LAMC2 genes has been shown to decrease the expression of these genes in prostate and breast cancer [65,66].

  • Identification of a novel family of laminin N-terminal alternate splice isoforms: Structural and functional characterization

    2009, Journal of Biological Chemistry
    Citation Excerpt :

    In contrast, LAMA3B contains exons 1–38 and 40–76 (i.e. skips exon 39), and encodes a full-length laminin termed α3b (Fig. 1B). Further alternate or minor isoforms have been identified in LAMC2 (18, 19), LAMA4 (20), LAMB3 (21), and LAMA2 (22). Here, we describe the identification of multiple, short, alternate splice isoforms derived from the 5′-end of the genes (LAMA3 and LAMA5) encoding the α3 and α5 laminin subunits.

  • Laminin isoforms containing the γ3 chain are unable to bind to integrins due to the absence of the glutamic acid residue conserved in the C-terminal regions of the γ1 and γ2 chains

    2008, Journal of Biological Chemistry
    Citation Excerpt :

    Construction of Expression Vectors—Soluble clasped α6, α7X2, and β1 integrin expression vectors were prepared as described previously (10, 33). Expression vectors for the human laminin α1, α3, α4, α5, β1, β3, and γ1 chains were constructed as described (16, 20, 33, 34). Expression vectors for the human laminin α2 (GenBank™ accession number NM_000426), β2 (GenBank™ accession number NM_002292), and γ3 (GenBank™ accession number NM_006059) chains were prepared as follows.

  • Recombinant human laminin isoforms can support the undifferentiated growth of human embryonic stem cells

    2008, Biochemical and Biophysical Research Communications
    Citation Excerpt :

    Preparation of culture vessels coated with recombinant laminins. Five recombinant human laminin isoforms, rhLM-111, -211, -332, -411, and -511 were produced using the Free Style™ 293 Expression System (Invitrogen) and purified from conditioned medium as described previously [14,15]. The rhLMs were filtered through the 0.45-μm pores of Ultrafree-CL Centrifugal Filter Units (MILLIPORE) for sterile usage.

  • The requirement of the glutamic acid residue at the third position from the carboxyl termini of the laminin γ chains in integrin binding by laminins

    2007, Journal of Biological Chemistry
    Citation Excerpt :

    αV and β3 integrin expression vectors were generously provided by Dr. Junichi Takagi (31, 32). Expression vectors for human laminin α5 chain lacking LG4-5 modules (pcDNA-α5ΔLG4-5), LG3–5 modules (pcDNA-α5ΔLG3–5), β1 chain (pCEP-β1), and γ1 chain (pcDNA3.1-γ1) were constructed as described (19, 33). Expression vectors for the laminin γ1 chain lacking the C-terminal 8 amino acids (γ1Δ8AA), 3 amino acids (γ1Δ3AA), 2 amino acids (γ1Δ2AA), or 1 amino acid (γ1Δ1AA) and that encoding the mutant γ1 chain with substitution of Gln for Glu-1607 (γ1EQ) were prepared as follows.

View all citing articles on Scopus
View full text